Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same

ABSTRACT

Methods for synthesizing proinsulin polypeptides are described that include a contacting a proinsulin polypeptide including an insulin polypeptide coupled to one or more peptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate, and cleaving the one or more peptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. Methods of synthesizing proinsulin polypeptide-oligomer conjugates are also described as are proinsulin polypeptide-oligomer conjugates. Methods of synthesizing C-peptide polypeptide-oligomer conjugates are also described.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/318,197, filed Sep. 7, 2001, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to insulin conjugates, methods ofsynthesizing such conjugates, and methods of treating diseases includingdiabetes therewith.

BACKGROUND OF THE INVENTION

The polypeptide insulin is the primary hormone responsible forcontrolling the transport, utilization and storage of glucose in thebody. The β-cells of the pancreatic islets secrete a single chainprecursor of insulin, known as proinsulin. Proteolysis of proinsulinresults in removal of certain basic amino acids in the proinsulin chainand the connecting or C-peptide and provides the biologically activepolypeptide insulin.

The insulin molecule has been highly conserved in evolution andgenerally consists of two chains of amino acids linked by disulfidebonds. In the natural human, two-chain insulin molecule (mw 5,800Daltons), the A-chain is composed of 21 amino acid residues and hasglycine at the amino terminus; and the B-chain has 30 amino acidresidues and phenylalanine at the amino terminus.

Insulin may exist as a monomer or may aggregate into a dimer or ahexamer formed from three of the dimers. Biological activity, i.e., theability to bind to receptors and stimulate the biological actions ofinsulin, resides in the monomer.

Diabetes is a biological disorder involving improper carbohydratemetabolism. Diabetes results from insufficient production of or reducedsensitivity to insulin. In persons with diabetes, the normal ability touse glucose is inhibited, thereby increasing blood sugar levels(hyperglycemia). As glucose accumulates in the blood, excess levels ofsugar are excreted in the urine (glycosuria). Other symptoms of diabetesinclude increased urinary volume and frequency, thirst, itching, hunger,weight loss, and weakness.

There are two varieties of diabetes. Type I is insulin-dependentdiabetes mellitus, or IDDM. IDDM was formerly referred to as “juvenileonset diabetes.” In IDDM, insulin is not secreted by the pancreas andmust be provided from an external source. Type II or adult-onsetdiabetes can ordinarily be controlled by diet, although in some advancedcases insulin is required.

Before the isolation of insulin in the 1920s, most patients died withina short time after onset. Untreated diabetes leads to ketosis, theaccumulation of ketones, products of fat breakdown, in the blood. Thisis followed by the accumulation of acid in the blood (acidosis) withnausea and vomiting. As the toxic products of disordered carbohydrateand fat metabolism continue to build up, the patient goes into adiabetic coma, which leads to death.

The use of insulin as a treatment for diabetes dates to 1922, whenBanting et al. (“Pancreatic Extracts in the Treatment of DiabetesMellitus,” Can. Med. Assoc. J., 12:141-146 (1922)) showed that theactive extract from the pancreas had therapeutic effects in diabeticdogs. In that same year, treatment of a diabetic patient with pancreaticextracts resulted in a dramatic, life-saving clinical improvement.

Until recently, bovine and porcine insulin were used almost exclusivelyto treat diabetes in humans. Today, however, numerous variations ininsulin between species are known. Each variation differs from naturalhuman insulin in having amino acid substitution(s) at one or morepositions in the A- and/or B-chain. Despite these differences, mostmammalian insulin has comparable biological activity. The advent ofrecombinant technology allows commercial scale manufacture of humaninsulin (e.g., Humulin™ insulin, commercially available from Eli Lillyand Company, Indianapolis, Ind.) or genetically engineered insulinhaving biological activity comparable to natural human insulin.

Treatment of diabetes typically requires regular injections of insulin.Due to the inconvenience of insulin injections, massive efforts toimprove insulin administration and bioassimilation have been undertaken.

Attempts have been made to deliver insulin by oral administration. Theproblems associated with oral administration of insulin to achieveeuglycemia in diabetic patients are well documented in pharmaceuticaland medical literature. Digestive enzymes in the gastrointestinal tractrapidly degrade insulin, resulting in biologically inactive breakdownproducts. In the stomach, for example, orally administered insulinundergoes enzymatic proteolysis and acidic degradation. Comparableproteolytic breakdown of insulin occurs in the intestine. In the lumen,insulin is attacked by a variety of enzymes including gastric andpancreatic enzymes, exo- and endopeptidases, and brush borderpeptidases. Even if insulin survives this enzymatic attack, thebiological barriers that must be traversed before insulin can reach itsreceptors in vivo may limit its bioavailability after oraladministration of insulin. For example, insulin may possess low membranepermeability, limiting its ability to pass from the intestinal lumeninto the bloodstream.

Some efforts to provide an oral form of insulin have focused onproviding insulin-oligomer conjugates. Human insulin and many closelyrelated insulins that are used therapeutically contain three amino acidresidues bearing free primary amino groups. All three primary aminogroups, namely the N-termini (alpha amino groups) of the A and B chains(Gly^(A1) and Phe^(B1)) and the epsilon-ammo group of Lys^(B29), may bemodified by conjugation with oligomers. Depending on the reactionconditions, N-acylation of an unprotected insulin leads to a complexmixture of mono-, di-, and tri-conjugates (e.g., insulin mono-conjugatedat Gly ^(A1), insulin mono-conjugated at Phe^(B1), insulinmono-conjugated at Lys^(B29), insulin conjugated at Gly^(A1) andPhe^(B1), insulin di-conjugated at Gly^(A1) and Lys^(B29), insulindi-conjugated at Phe^(B1) and Lys^(B29), and insulin tri-conjugated atGly^(A1), Phe^(B1), and Lys^(B29)). When a particular conjugate, forexample insulin mono-conjugated at Lys^(B29), is desired, it can beburdensome and/or expensive to separate (or purify) such a complexmixture of conjugates to obtain the desired conjugate.

As a result, various efforts have been undertaken to selectivelysynthesize the desired insulin conjugate. For example, Muranishi andKiso, in Japanese Patent Application 1-254,699, propose a five-stepsynthesis for preparing fatty acid insulin derivatives. The A1- and B1-amino groups of insulin are protected (or blocked) withp-methoxybenzoxy carbonyl azide (pMZ). After acylation with a fatty acidester, the protection (blocking) groups are removed to provide insulinmono-acylated at Lys(B29) with a fatty acid. As another example, U.S.Pat. No. 5,750,497 to Havelund et al. proposes treating human insulinwith a Boc-reagent (e.g. di-tert-butyl dicarbonate) to form(A1,B1)-diBoc human insulin, i.e., human insulin in which the N-terminalend of both the A- and B-chains are protected by a Boc-group. After anoptional purification, e.g. by HPLC, a lipophilic acyl group isintroduced in the &-amino group of Lys^(B29) by allowing the product toreact with a N-hydroxysuccinimide ester of the formula X-OSu wherein Xis the lipophilic acyl group to be introduced. In the final step,trifluoroacetic acid is used to remove the Boc-groups and the product,N^(εB29)-X human insulin, is isolated.

Various other efforts have been undertaken to preferentially synthesizethe desired insulin conjugate to provide a mixture of conjugates inwhich the desired insulin conjugate is the preferred product. Forexample, U.S. Pat. No. 5,646,242 to Baker et al. proposes a reactionthat is performed without the use of amino-protecting groups. Bakerproposes the reaction of an activated fatty ester with the &-amino groupof insulin under basic conditions in a polar solvent. The acylation ofthe ε-amino group is dependent on the basicity of the reaction. At a pHgreater than 9.0, the reaction preferentially acylates the ε-amino groupof B29-lysine over the α-amino groups. Examples 1 through 4 reportreaction yields of the mono-conjugated insulin as a percentage of theinitial amount of insulin between 67.1% and 75.5%. In Example 5, Bakeralso proposes acylation of human proinsulin with N-succinimidylpalmitate. The exact ratios of ε-amino acylated species to α-aminoacylated species were not calculated. The sum of all ε-amino acylatedspecies within the chromatogram accounted for 87-90% of the total area,while the sum of all related substances (which would presumably includeany α-amino acylated species) accounted for <7% of the total area, forany given point in time.

It is desirable to provide methods of site specifically synthesizingdesired, particular insulin-oligomer conjugates that may be lessburdensome and/or more cost effective than the conventional methodsdescribed above.

SUMMARY OF THE INVENTION

When compared to the conventional schemes described above, embodimentsof the present invention may provide a commercially less expensiveand/or higher yielding manufacturing scheme for producinginsulin-oligomer conjugates where site-specific conjugation is desirable(e.g., where it is desirable to provide an insulin mono-conjugate havingthe oligomer coupled to the B-29 Lys of the insulin molecule). Unlikethe conventional schemes described above, which propose selectiveconjugation of insulin by blocking the N-termini of the insulin withcompounds such as p-methoxybenzoxy carbonyl azide (Muranishi and Kiso)or by attempting to control the reaction conditions to reduce but noteliminate conjugation at the N-termini of the insulin (Baker),embodiments of the present invention couple the oligomer to the B-29 Lysof proinsulin or of artificial proinsulin (e.g., proinsulin coupled atthe N-terminus of its B-chain to a leader peptide). The C-peptide (andleader peptide, if present) is then cleaved from the proinsulin-oligomerconjugate to provide insulin mono-conjugated at B-29 Lys with theoligomer. Embodiments of the present invention may provide high sitespecificity for B-29 Lys modification. Methods according to embodimentsof the present invention utilizing proinsulin polypeptides may providehigh conversion B-29 modified product, for example with yields as highas 80% or greater, compared with that obtained via conventional insulinpathways.

According to embodiments of the present invention, a method ofsynthesizing an insulin polypeptide-oligomer conjugate includescontacting a proinsulin polypeptide comprising an insulin polypeptidecoupled to one or more peptides by peptide bond(s) capable of beingcleaved to yield the insulin polypeptide with an oligomer underconditions sufficient to couple the oligomer to the insulin polypeptideportion of the proinsulin polypeptide and provide a proinsulinpolypeptide-oligomer conjugate, and cleaving the one or more peptidesfrom the proinsulin polypeptide-oligomer conjugate to provide theinsulin polypeptide-oligomer conjugate.

According to other embodiments of the present invention, a method ofsynthesizing an insulin polypeptide-acyl oligomer conjugate comprisingenzymatically cleaving one or more peptides from a proinsulinpolypeptide-acyl oligomer conjugate to provide the insulinpolypeptide-acyl oligomer conjugate.

According to other embodiments of the present invention, a method ofsynthesizing a proinsulin polypeptide-oligomer conjugate includescontacting a proinsulin polypeptide comprising an insulin polypeptidecoupled to one or more peptides by peptide bond(s) capable of beingcleaved to yield the insulin polypeptide with an oligomer underconditions sufficient to couple the oligomer to the insulin polypeptideportion of the proinsulin polypeptide and provide the proinsulinpolypeptide-oligomer conjugate.

According to still other embodiments of the present invention, aproinsulin polypeptide-oligomer conjugate includes a proinsulinpolypeptide including an insulin polypeptide, and an oligomer coupled tothe insulin polypeptide portion of the proinsulin polypeptide.

According to yet other embodiments of the present invention, a method ofsynthesizing a C-peptide polypeptide-oligomer conjugate includescontacting a pro-C-peptide polypeptide comprising a C-peptidepolypeptide coupled to one or more peptides by peptide bond(s) that arecleavable to yield the C-peptide polypeptide with an oligomer underconditions sufficient to couple the oligomer to the C-peptidepolypeptide portion of the pro-C-peptide polypeptide and provide apro-C-peptide polypeptide-oligomer conjugate, and cleaving the one ormore peptides from the pro-C-peptide polypeptide-oligomer conjugate toprovide the C-peptide polypeptide-oligomer conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates embodiments of a synthesis route for preparation ofB-29 Lys modified insulin using a proinsulin having a leader peptide;

FIG. 2 illustrates an HPLC profile of proinsulin II conjugation;

FIG. 3 illustrates an m.s. spectrum of purified proinsulin IImonoconjugate;

FIG. 4 illustrates an m.s. spectrum of purified proinsulin IIdiconjugate;

FIG. 5 illustrates an HPLC profile of production of Insulin-hexyl-PEG7monoconjugate;

FIG. 6 illustrates an m.s. spectrum of the product of trypsin cleavageof a proinsulin II monoconjugate;

FIG. 7 illustrates an HPLC profile of Insulin(Arg³¹)-hexyl-PEG7 cleavageby carboxy peptidase B;

FIG. 8 illustrates an m.s. spectrum of the product of carboxypeptidasecleavage of B-29 acylated Insulin(Arg³¹)hexyl-PEG7 conjugate;

FIG. 9 illustrates an HPLC profile of the production ofInsulin-hexyl-PEG7 (polydispersed) from Proinsulin II monoconjugatecleaved by an enxyme cocktail of carboxy peptidase B and trypsin;

FIG. 10 illustrates an m.s. spectrum of Insulin-hexyl-PEGn(polydispersed) via proinsulin II;

FIG. 11 illustrates an HPLC profile of production of B-29 acylatedInsulin-hexyl-PEG7 via proinsulin 1;

FIG. 12 illustrates an m.s. spectrum of B-29 acylated Insulin-hexyl-PEG7via proinsulin I;

FIG. 13 illustrates an m.s. spectrum Of insulin (side product) derivedfrom proinsulin I conjugate mixture;

FIG. 14 illustrates an HPLC profile of proinsulin I monoconjugate A,proinsulin I monoconjugate B and proinsulin I diconjugate;

FIG. 15 illustrates an HPLC profile of production of Insulin-hexyl-PEG7monoconjugate from reaction of proinsulin I diconjugate with enzymecocktail of carboxy peptidase B and trypsin; and

FIG. 16 illustrates an HPLC profile of production of insulin (sideproduct) from reaction of proinsulin I monoconjugate A with enzymecocktail of carboxy peptidase B and trypsin.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

All amino acid abbreviations used in this disclosure are those acceptedby the United States Patent and Trademark Office as set forth in 37C.F.R. § 1.822(b).

As used herein, the term “between” when used to describe various rangesshould be interpreted to include the end-points of the described ranges.

As used herein, the term “substantially monodispersed” is used todescribe a mixture of compounds wherein at least about 95 percent of thecompounds in the mixture have the same molecular weight.

As used herein, the term “monodispersed” is used to describe a mixtureof compounds wherein about 100 percent of the compounds in the mixturehave the same molecular weight.

As used herein, the term “insulin polypeptide” means a polypeptidepossessing at least some of the biological activity of insulin (e.g.,ability to affect the body through insulin's primary mechanism ofaction). For example, an insulin polypeptide may be a polypeptide suchas insulin having an A-chain polypeptide and a B-chain polypeptidecoupled to the A-chain polypeptide by disulfide bonds. In variousembodiments of the present invention, the insulin polypeptide preferablypossesses a majority of the biological activity of insulin, morepreferably possesses substantially all of the biological activity ofinsulin, and most preferably possesses all of the biological activity ofinsulin.

As used herein, the term “proinsulin polypeptide” means an insulinpolypeptide that is coupled to one or more peptides (e.g., leaderpeptides and/or connecting or C-peptides) by peptide bond(s) that arecapable of cleavage in vitro or in vivo. For example, a proinsulinpolypeptide may include an insulin polypeptide, such as insulin, havingan A-chain polypeptide coupled to a B-chain polypeptide by bonds such asdisulfide bonds, and a connecting peptide coupled to the C-terminus ofthe B-chain polypeptide and coupled to the N-terminus of the A-chainpolypeptide by peptide bonds that are capable of cleavage in vitroand/or in vivo. As another example, a proinsulin polypeptide may includean insulin polypeptide, such as insulin, having an A-chain polypeptidecoupled to a B-chain polypeptide by bonds such as disulfide bonds, aconnecting peptide coupled to the C-terminus of the B-chain polypeptideand coupled to the N-terminus of the A-chain polypeptide by peptidebonds that are capable of cleavage in vitro and/or in vivo, and a leaderpeptide coupled to the N-terminus of the B-chain polypeptide. Exemplaryproinsulin polypeptides include, but are not limited to, proinsulin,proinsulin analogs, proinsulin fragments, proinsulin analog fragments,or any of proinsulin, proinsulin analogs, proinsulin fragments,proinsulin analog fragments having a leader peptide; preproinsulin,preproinsulin anaolgs, preproinsulin fragments, preproinsulin fragmentanalogs, miniproinsulin, and fusion proteins.

As used herein, the term “insulin” means the insulin of one of thefollowing species: human, cow, pig, sheep, horse, dog, chicken, duck orwhale, provided by natural, synthetic, or genetically engineeredsources. In various embodiments of the present invention, insulin ispreferably human insulin.

As used herein, the term “insulin analog” means insulin wherein one ormore of the amino acids have been replaced while retaining some or allof the activity of the insulin. The analog is described by noting thereplacement amino acids with the position of the replacement as asuperscript followed by a description of the insulin. For example,“Pro^(B29) insulin, human” means that the lysine typically found at theB29 position of a human insulin molecule has been replaced with proline.

Insulin analogs may be obtained by various means, as will be understoodby those skilled in the art. For example, certain amino acids may besubstituted for other amino acids in the insulin structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. As the interactive capacity and nature ofinsulin defines its biological functional activity, certain amino acidsequence substitutions can be made in the amino acid sequence andnevertheless remain a polypeptide with like properties.

In making such substitutions, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is accepted that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant polypeptide, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics as follows: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5). As will beunderstood by those skilled in the art, certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity, i.e., still obtain a biological functionally equivalentpolypeptide. In making such changes, the substitution of amino acidswhose hydropathic indices are within ±2 of each other is preferred,those which are within ±1 of each other are particularly preferred, andthose within ±0.5 of each other are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, the disclosure of which is incorporate herein in itsentirety, provides that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (±3.0);aspartate (+3.0±1); glutamate (+3.0±1); seine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4). As is understood by thoseskilled in the art, an amino acid can be substituted for another havinga similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalentpolypeptide. In such changes, the substitution of amino acids whosehydrophilicity values are within 2 of each other is preferred, thosewhich are within ±1 of each other are particularly preferred, and thosewithin ±0.5 of each other are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions (i.e., amino acids that maybe interchanged without significantly altering the biological activityof the polypeptide) that take various of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, for example: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

As will be understood by those skilled in the art, insulin analogs maybe prepared by a variety of recognized peptide synthesis techniquesincluding, but not limited to, classical (solution) methods, solid phasemethods, semi-synthetic methods, and recombinant DNA methods.

Examples of human insulin analogs include, but are not limited to,Gly^(A21) insulin, human; Gly^(A21) Gln^(B3) insulin, human; Ala^(A21)insulin, human; Ala^(A21) Gln^(B3) insulin, human; Gln^(B3) insulin,human; Gln^(B30) insulin, human; Gly^(A21) Glu^(B30) insulin, human;Gly^(A21) Gln^(B3) Glu^(B30) insulin, human; Gln^(B30) Glu^(B30)insulin, human; Asp^(B28) insulin, human; Lys^(B28) insulin, human;Leu^(B28) insulin, human; Val^(B28) insulin, human; Ala^(B28) insulin,human; Asp^(B28) pro^(B29) insulin, human; Lys^(B28) Pro^(B29) insulin,human; Leu^(B28) Pro^(B29) insulin, human; Val^(B28) pro^(B29) insulin,human; Ala^(B28) Pro^(B29) insulin, human.

As used herein, the term “insulin fragment” means a segment of the aminoacid sequence found in the insulin that retains some or all of theactivity of the insulin. Insulin fragments are denoted by stating theposition(s) in an amino acid sequence followed by a description of theamino acid. For example, a “B25-B30 human insulin” fragment would be thesix amino acid sequence corresponding to the B25, B26, B27, B28, B29 andB30 positions in the human insulin amino acid sequence.

As used herein, the term “insulin fragment analog” means a segment ofthe amino acid sequence found in the insulin molecule wherein one ormore of the amino acids in the segment have been replace while retainingsome or all of the activity of the insulin.

As used herein, the term “proinsulin” means the proinsulin of one of thefollowing species: human, cow, pig, sheep, horse, dog, chicken, duck orwhale, provided by natural, synthetic, or genetically engineeredsources. In general, proinsulin consist of insulin having a C-peptideconnecting the N-terminus of the A chain of the insulin to theC-terminus of the B chain of the insulin. In various embodiments of thepresent invention described herein, the proinsulin is preferably humanproinsulin.

As used herein, the term “proinsulin analog” means proinsulin whereinone or more of the amino acids in proinsulin have been replaced asdescribed above with respect to insulin analogs while retaining some orall of the activity of the insulin portion of the proinsulin. The analogis described by noting the replacement amino acids with the position ofthe replacement as a superscript followed by a description of theproinsulin. For example, “Pro^(B29) proinsulin, human” means that thelysine typically found at the B29 position of a human proinsulinmolecule has been replaced with proline.

As used herein, the term “proinsulin fragment” means a segment of theamino acid sequence found in the proinsulin that retains some or all ofthe biological activity of the insulin, insulin analog or insulinfragment portion of the proinsulin fragment. Proinsulin fragments aredenoted by stating the position(s) in an amino acid sequence followed bya description of the amino acid. For example, a “B25-B35 humanproinsulin” fragment would be the eleven amino acid sequencecorresponding to the B25, B26, B27, B28, B29, B30, B31, B32, B33, B34and B35 positions in the human proinsulin amino acid sequence.

As used herein, the term “proinsulin fragment analog” means a segment ofthe amino acid sequence found in proinsulin molecule wherein one or moreof the amino acids in the segment have been replaced as described abovewith reference to insulin analogs while retaining some or all of theactivity of the insulin, insulin analog, insulin fragment, or insulinfragment analog portion of the proinsulin fragment.

As used herein, the term “preproinsulin” means the preproinsulin of oneof the following species: human, cow, pig, sheep, horse, dog, chicken,duck or whale, provided by natural, synthetic, or genetically engineeredsources. In general, preproinsulin is a single chain polypeptide (e.g.,a polypeptide having a leader peptide coupled to the N-terminus of theB-chain of the insulin and having the C-terminus of the B-chain coupledto the N-terminus of the A-chain by a connecting peptide) in which theA-chain is coupled to the B-chain by, for example, disulfide bonds. Inthe various embodiments of the present invention described herein, thepreproinsulin is preferably human preproinsulin.

As used herein, the term “preproinsulin analog” means preproinsulinwherein one or more of the amino acids in preproinsulin have beenreplaced as described above with respect to insulin analogs whileretaining some or all of the activity of the insulin or insulin analogportion of the preproinsulin analog. The analog is described by notingthe replacement amino acids with the position of the replacement as asuperscript followed by a description of the insulin.

As used herein, the term “preproinsulin fragment” means a segment of theamino acid sequence found in preproinsulin that retains some or all ofthe biological activity of the insulin or insulin fragment portion ofthe preproinsulin fragment. Preproinsulin fragments are denoted bystating the position(s) in an amino acid sequence followed by adescription of the amino acid.

As used herein, the term “preproinsulin fragment analog” means a segmentof the amino acid sequence found in preproinsulin molecule wherein oneor more of the amino acids in the segment have been replaced asdescribed above with reference to insulin analogs while retaining someor all of the activity of the insulin, insulin analog, insulin fragmentor insulin fragment analog portion of the preproinsulin fragment analog.

As used herein, the term “miniproinsulin” refers to a single-chaininsulin propolypeptide having an A-chain polypeptide and a B-chainpolypeptide, where the N- or C-terminus of the A-chain is coupled to theC- or N-terminus of the B-chain by a connecting peptide having between alower limit of 1, 2, 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid residues, and wherein the A-chainpolypeptide is coupled to the B-chain polypeptide by bonds, such asdisulfide bonds. Miniproinsulins may be various miniproinsulins as willbe understood by those skilled in the art including, but not limited to,those described in U.S. Pat. No. 5,157,021 to Balschmidt et al. and U.S.Pat. No. 5,202,415 to Jonassen et al., the disclosures of each of whichare incorporated by reference herein in their entireties.

As used herein, the term “C-peptide” means a peptide having the aminoacid sequence of the C-peptide of the proinsulin of one of the followingspecies: human, monkey, cow, pig, sheep, horse, dog, chicken, duck orwhale, provided by natural, synthetic, or genetically engineeredsources. In various embodiments of the present invention describedherein, the C-peptide is preferably human C-peptide.

As used herein, the term “C-peptide analog” means C-peptide wherein oneor more of the amino acids in the C-peptide have been replaced asdescribed above with respect to insulin analogs while retaining some orall of the biological activity of the C-peptide. Preferably, theC-peptide analog comprises the pentapeptide segment at the C-terminus ofa C-peptide and/or the nonapeptide segment found at positions 11-19 of aC-peptide. When the C-peptide analog comprises the pentapeptide segment,the pentapeptide segment is preferably at the C-terminus of theC-peptide analog. More preferably, the C-peptide analog comprises thetetrapeptide segment at the C-terminus of a C-peptide and/or thenonapeptide segment found at positions 11-19 of a C-peptide. When theC-peptide analog comprises the tetrapeptide segment, the tetrapeptidesegment is preferably at the C-terminus of the C-peptide analog. Thenonapeptide segment found at positions ll-19 of a C-peptide describedabove is preferably the nonapeptide segment fount at positions 11-19 ofhuman C-peptide.

As used herein, the term “C-peptide fragment” means a segment of theamino acid sequence of C-peptide that retains some, substantially all,or all of the biological activity of the C-peptide. Preferably, theC-peptide fragment comprises the pentapeptide segment at the C-terminusof a C-peptide and/or the nonapeptide segment found at positions 11-19of a C-peptide. When the C-peptide fragment comprises the pentapeptidesegment, the pentapeptide segment is preferably at the C-terminus of theC-peptide fragment. More preferably, the C-peptide fragment comprisesthe tetrapeptide segment at the C-terminus of a C-peptide and/or thenonapeptide segment found at positions 11-19 of a C-peptide. When theC-peptide fragment comprises the tetrapeptide segment, the tetrapeptidesegment is preferably at the C-terminus of the C-peptide fragment Stillmore preferably, the C-peptide fragment consists of a peptide selectedfrom the group consisting of the petapetide segment of the C-terminus ofa C-peptide, the nonapeptide segment found at positions 11-19 of aC-peptide, and the terapeptide segment of the C-terminus of a C-peptide.The nonapeptide segment found at positions 11-19 of a C-peptidedescribed above is preferably the nonapeptide segment fount at positions11-19 of human C-peptide.

As used herein, the term “C-peptide fragment analog” means a segment ofthe amino acid sequence of C-peptide wherein one or more of the aminoacids in the segment have been replaced as described above withreference to insulin analogs while retaining some, substantially all, orall of the biological activity of the insulin. Preferably, the C-peptidefragment analog comprises the pentapeptide segment at the C-terminus ofa C-peptide and/or the nonapeptide segment found at positions 11-19 of aC-peptide. When the C-peptide fragment analog comprises the pentapeptidesegment, the pentapeptide segment is preferably at the C-terminus of theC-peptide fragment analog. More preferably, the C-peptide fragmentanalog comprises the tetrapeptide segment at the C-terminus of aC-peptide and/or the nonapeptide segment found at positions 11-19 of aC-peptide. When the C-peptide fragment analog comprises the tetrapeptidesegment, the tetrapeptide segment is preferably at the C-terminus of theC-peptide fragment analog. The nonapeptide segment found at positions11-19 of a C-peptide described above is preferably the nonapeptidesegment fount at positions 11-19 of human C-peptide.

As used herein, the term “C-peptide polypeptide” means a polypeptidehaving a therapeutic utility and biological activity similar to thetherapeutic utility and biological 13functionality for C-peptides and/orC-peptide fragments described in J. Wahren et al., “Role of C-peptide inHuman Physiology,” Am. J. Physiol. Endocrinol. Metab., 278: E759-E768(2000) and/or T. Forst et al., “New Aspects on Biological Activity ofC-peptide in IDDM Patients,” Exp. Clin. Endocrinol. Diabetes, 106:270-276 (1998), the disclosures of which are incorporated herein byreference in their entireties. For example, C-peptide polypeptides havetherapeutic utility that includes, but is not limited to, decreasedglomerular hyperfiltration, augmented whole body and/or skeletal muscleglucose utilization, improved autonomic nerve function, and/or aredistribution of microvascular skin blood flow. C-peptide polypeptideshave biological activity that includes, but is not limited to, theability to stimulate Na⁺-K⁺-ATPase activity, the ability to stimulateendotheial nitric oxide synthase activity, and/or the ability to bindspecifically to cell surfaces (e.g., at a G-protein-coupled surfacereceptor) with subsequent activation of Ca²⁺-dependent intracellularsignaling pathways. C-peptide polypeptides preferably have anassociation rate constant for binding to endothelial cells, renaltubular cells, and fibroblasts of ˜3×10⁹ M⁻¹. C-peptide polypeptides arepreferably C-peptides, C-peptide analogs, C-peptide fragments, orC-peptide fragment analogs.

As used herein, the term “pro-C-peptide polypeptide” means a C-peptidepolypeptide coupled to one or more peptides that are cleavable toprovide the C-peptide polypeptide.

As used herein, the term “A-chain polypeptide” means a polypeptide thatis substantially biologically equivalent to the A-chain of an insulinmolecule. For example, A-chain polypeptides may be A-chain analogs,which may be provided as described above with respect to insulinanalogs, A-chain fragments, or A-chain analog fragments.

As used herein, the term “B-chain polypeptide” means a polypeptide thatis substantially biologically equivalent to the B-chain of an insulinmolecule. For example, B-chain polypeptides may be B-chain analogs,which may be provided as described above with respect to insulinanalogs, B-chain fragments, or B-chain analog fragments.

As used herein, the term “polypeptide” means a peptide having two ormore amino acid residues.

As used herein, the term “amphiphilically balanced” means capable ofsubstantially dissolving in water and capable of penetrating biologicalmembranes.

As used herein, the term “polyalkylene glycol” refers to straight orbranched polyalkylene glycol polymers such as polyethylene glycol,polypropylene glycol, and polybutylene glycol, and includes themonoalkylether of the polyalkylene glycol. The term “polyalkylene glycolsubunit” refers to a single polyalkylene glycol unit. For example, apolyethylene glycol subunit would be —O—CH₂—CH₂—O—.

As used herein, the term “lipophilic” means the ability to dissolve inlipids and/or the ability to penetrate, interact with and/or traversebiological membranes, and the term, “lipophilic moiety” or “lipophile”means a moiety which is lipophilic and/or which, when attached toanother chemical entity, increases the lipophilicity of such chemicalentity. Examples of lipophilic moieties include, but are not limited to,alkyls, fatty acids, esters of fatty acids, cholesteryl, adamantyl andthe like.

As used herein, the term “lower alkyl” refers to substituted orunsubstituted alkyl moieties having from one to five carbon atoms.

As used herein, the term “higher alkyl” refers to substituted orunsubstituted alkyl moieties having six or more carbon atoms.

According to embodiments of the present invention, methods ofsynthesizing an insulin polypeptide-oligomer conjugate includecontacting a proinsulin polypeptide comprising an insulin polypeptidecoupled to one or more peptides by peptide bond(s) capable of beingcleaved to yield the insulin polypeptide with an oligomer underconditions sufficient to couple the oligomer to the insulin polypeptideportion of the proinsulin polypeptide and provide a proinsulinpolypeptide-oligomer conjugate, and cleaving the one or more peptidesfrom the proinsulin polypeptide-oligomer conjugate to provide theinsulin polypeptide-oligomer conjugate. For example, insulin-oligomerconjugates may be synthesized as described in the Examples providedbelow. An embodiment of a synthesis route is provided in FIG. 1.

The proinsulin polypeptide may be various proinsulin polypeptidescomprising an insulin polypeptide coupled to one or more peptides bypeptide bond(s) capable of being cleaved to yield the insulinpolypeptide as will be understood by those skilled in the art including,but not limited to, proinsulin, proinsulin analogs, proinsulinfragments, proinsulin fragment analogs, miniproinsulin, or fusionproteins. In some embodiments, the proinsulin polypeptide is aproinsulin analog having a leader peptide. The proinsulin analog havinga leader peptide is produced by Itoham Foods, Inc. of Ibaraki Pref,Japan. The leader peptide and the C-peptide of the proinsulin analog areeach devoid of lysine residues. In other embodiments, the proinsulinpolypeptide is a proinsulin poplypeptide produced by Biobras of BeloHorizonte, Brazil. The proinsulin polypeptide has a leader peptidecoupled to the N-terminus of the B-chain of the proinsulin. The leaderpeptide is devoid of lysine residues.

The insulin polypeptide preferably has an A-chain polypeptide and aB-chain polypeptide. The A-chain polypeptide is preferably devoid oflysine residues. The B-chain polypeptide preferably comprises a singlelysine residue. The A-chain polypeptide and the B-chain polypeptide arepreferably cross-linked, and are more preferably cross-linked using oneor more disulfide bonds. Still more preferably, the A-chain polypeptideand the B-chain polypeptide each comprise cysteine residues, one or moreof which are coupled using one or more disulfide bonds to cross-link theA-chain polypeptide with the B-chain polypeptide. Preferably, theinsulin polypeptide is insulin, an insulin analog, an insulin fragment,or an insulin analog fragment.

In some embodiments, the one or more peptides coupled to the insulinpolypeptide comprise a connecting peptide coupled at a first end to theC-terminus of the B-chain polypeptide and at a second end to theN-terminus of the A-chain polypeptide. In general, the amino acidsequence of the connecting peptide is not critical and the connectingpeptide may be various connecting peptides as will be understood bythose skilled in the art including, but not limited to, C-peptidepolypeptides, C-peptides, and the connecting peptides inminiproinsulins. In some embodiments, the connecting peptide is devoidof lysine residues. These embodiments may utilize less oligomericreagents by reducing the number of possible conjugation sites on theproinsulin polypeptide molecule.

In other embodiments, the one or more peptides coupled to the insulinpolypeptide comprise a leader peptide that is coupled to the N-terminusof the B-chain polypeptide. In general, the amino acid sequence of theleader peptide is not critical. In some embodiments, the leader peptideis devoid of lysine residues. These embodiments may reduce the amount ofoligomeric reagent used by limiting the number of conjugation sites onthe proinsulin polypeptide molecule.

In still other embodiments, the one or more peptides coupled to theinsulin polypeptide comprise both a connecting peptide as describedabove and a leader peptide as described above. The one or more peptidesmay consist essentially of a connecting peptide and a leader peptide, ormay consist of a connecting peptide and a leader peptide.

The peptide bonds are bonds that may be cleaved in various ways as willbe understood by those skilled in the art. Preferably, the peptide bondsare bonds that may be enzymatically cleaved by enzymes including, butnot limited to, trypsin, carboxy peptidase B, thrombin, pepsin, andchymotripsin. Peptide bonds that may be enzymatically cleaved will beunderstood by those skilled in the art and include, but are not limitedto, Arg-Arg, Thr-Arg, Ala-Arg, Thr-Arg-Arg, Thr-Lys, Arg-Gly, andArg-Phe.

The oligomer may be various oligomers as will be understood by thoseskilled in the art. In general, the oligomer may be any oligomer capableof being coupled to a polypeptide as will be understood by those skilledin the art. For example, the oligomer may be a poly-dispersed oligomeras described in U.S. Pat. No. 4,179,337 to Davis et al.; U.S. Pat. No.5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe; U.S. Pat.No. 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 to Ekwuribe, and U.S.Pat. No. 6,309,633 to Ekwuribe et al., the disclosures of each of whichare incorporated herein by reference in their entireties. As anotherexample, the oligomer may be a non-polydispersed oligomer as describedin U.S. patent application Ser. No. 09/873,731 filed Jun. 4, 2001 byEkwuribe et al. entitled “Methods of Synthesizing SubstantiallyMonodispersed Mixtures of Polymers Having Polyethylene Glycol Mixtures”;U.S. patent application Ser. No. 09/873,797 filed Jun. 4, 2001 byEkwuribe et al. entitled “Mixtures of Drug-Oligomer ConjugatesComprising Polyalkylene Glycol, Uses Thereof, and Methods of MakingSame”; and U.S. patent application Ser. No. 09/873,899 filed Jun. 4,2001 by Ekwuribe et al. entitled “Mixtures of Insulin Drug-OligomerConjugates Comprising Polyalkylene Glycol, Uses Thereof, and Methods ofMaking Same,” the disclosures of each of which are incorporated hereinin their entireties.

In some embodiments, the oligomer comprises a hydrophilic moiety as willbe understood by those skilled in the art including, but not limited to,polyalkylene glycols such as polyethylene glycol or polypropyleneglycol, polyoxyethylenated polyols, copolymers thereof and blockcopolymers thereof, provided that the hydrophilicity of the blockcopolymers is maintained. The hydrophilic moiety is preferably apolyalkylene glycol moiety. The polyalkylene glycol moiety has at least1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. Thepolyalkylene glycol moiety more preferably has between a lower limit of2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 polyalkylene glycol subunits. Even morepreferably, the polyalkylene glycol moiety has between a lower limit of3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, or 12polyalkylene glycol subunits. The polyalkylene glycol moiety still morepreferably has between a lower limit of 4, 5, or 6 and an upper limit of6, 7, or 8 polyalkylene glycol subunits. The polyalkylene glycol moietymost preferably has 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety of the oligomer is preferably a lower alkyl polyalkyleneglycol moiety such as a polyethylene glycol moiety, a polypropyleneglycol moiety, or a polybutylene glycol moiety. When the polyalkyleneglycol moiety is a polypropylene glycol moiety, the moiety preferablyhas a uniform (i.e., not random) structure. An exemplary polypropyleneglycol moiety having a uniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

The oligomer may further comprise one or more additional hydrophilicmoieties (i.e., moieties in addition to the polyalkylene glycol moiety)including, but not limited to, sugars, polyalkylene glycols, andpolyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will beconsidered to be the same moiety if they are coupled by ether bonds. Forexample, the moiety—O—C₂H₄—O—C₂ H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—a single polyethylene glycol moiety having six polyethylene glycolsubunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

is a polyethylene glycol moiety having four polyethylene glycol subunitsand an additional hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and no additionalhydrophilic moieties.

The oligomer preferably further comprises one or more lipophilicmoieties as will be understood by those skilled in the art. Thelipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbon atoms. Thelipophilic moiety preferably has between a lower limit of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. The lipophilicmoiety more preferably has between a lower limit of 2, 3, 4, 5, 6, 7, 8,9, or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilic moiety even morepreferably has between a lower limit of 3, 4, 5, 6, 7, 8, or 9 and anupper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thelipophilic moiety still more preferably has between a lower limit of 3,4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10 carbon atoms. Thelipophilic moiety most preferably has 6 carbon atoms. The lipophilicmoiety is preferably selected from the group consisting of saturated orunsaturated, linear or branched alkyl moieties, saturated orunsaturated, linear or branched fatty acid moieties, cholesterol, andadamantane. Exemplary alkyl moieties include, but are not limited to,saturated, linear alkyl moieties such as methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl;saturated, branched alkyl moieties such as isopropyl, sec-butyl,tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derivedfrom the above saturated alkyl moieties including, but not limited to,vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary fatty acid moieties include, but are not limited to,unsaturated fatty acid moieties such as lauroleate, myristoleate,palmitoleate, oleate, elaidate, erucate, linoleate, linolenate,arachidonate, eicosapentaentoate, and docosahexaenoate; and saturatedfatty acid moieties such as acetate, caproate, caprylate, caprate,laurate, arachidate, behenate, lignocerate, and cerotate.

The oligomer may further comprise one or more spacer moieties as will beunderstood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theproinsulin polypeptide, to separate a first hydrophilic or lipophilicmoiety from a second hydrophilic or lipophilic moiety, or to separate ahydrophilic moiety or lipophilic moiety from a linker moiety. Spacermoieties are preferably selected from the group consisting of sugar,cholesterol and glycerine moieties. Sugar moieties may be various sugarmoieties as will be understood by those skilled in the art including,but not limited to, monosaccharide moieties and disaccharide moieties.Preferred monosaccharide moieties have between 4 and 6 carbon atoms.

The oligomer may further comprise one or more linker moieties that areused to couple the oligomer with the proinsulin polypeptide as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties. Thealkyl linker moiety may be a saturated or unsaturated, linear orbranched alkyl moiety as will be understood by those skilled in the artincluding, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Thealkoxy moiety may be various alkoxy moieties including, but not limitedto, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradocyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy,eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy,tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4, or 5and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moiety maybe a saturated or unsaturated, linear or branched fatty acid moiety aswill be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

The oligomer may further comprise one or more terminating moieties atthe one or more ends of the oligomer, which are not coupled to theinsulin polypeptide. The terminating moiety is preferably an alkyl oralkoxy moiety. The alkyl or alkoxy moiety preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbonatoms. The alkyl or alkoxy moiety more preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, or 7 and an upper limit of 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 carbon atoms. The alkyl or alkoxy moiety even morepreferably has between a lower limit of 1, 2, 3, 4, or 5 and an upperlimit of 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl or alkoxy moietystill more preferably has between a lower limit of 1, 2, 3, or 4 and anupper limit of 5, 6, or 7 carbon atoms. The alkyl moiety may be a linearor branched, saturated or unsaturated alkyl moiety as will be understoodby those skilled in the art including, but not limited to, methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl,2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl,ethynyl, 1-propynyl, and 2-propynyl. The alkoxy moiety may be variousalkoxy moieties including, but not limited to, methoxy, ethoxy, propoxy,butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy,sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy,2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The terminating moiety ismore preferably a lower alkyl moiety such as methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl, or alower alkoxy moiety such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. Mostpreferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

According to other embodiments of the present invention, the oligomercomprises the structure of Formula I:A-L_(j)-G_(k)-R-G′_(m)-R′-G″_(n)-T  (I)wherein:

-   -   A is an activatable moiety;    -   L is a linker moiety;    -   G, G′ and G″ are individually selected spacer moieties;

-   R is a lipophilic moiety and R′ is a polyalkylene glycol moiety, or    R′ is the lipophilic moiety and R is the polyalkylene glycol moiety;

-   T is a terminating moiety; and    -   j, k, m and n are individually 0 or 1.

According to these embodiments of the present invention, thepolyalkylene glycol moiety has at least 1, 2, 3, 4, 5, 6 or 7polyalkylene glycol subunits. The polyalkylene glycol moiety preferablyhas between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more polyalkylene glycol subunits. The polyalkylene glycol moietymore preferably has between a lower limit of 2, 3, 4, 5, or 6 and anupper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 polyalkylene glycol subunits. Even more preferably, the polyalkyleneglycol moiety has between a lower limit of 3, 4, 5, or 6 and an upperlimit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol subunits. Thepolyalkylene glycol moiety still more preferably has between a lowerlimit of 4, 5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycolsubunits. The polyalkylene glycol moiety most preferably has 7polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene glycol moiety is apolypropylene glycol moiety, the moiety preferably has a uniform (i.e.,not random) structure. An exemplary polypropylene glycol moiety having auniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

According to these embodiments of the present invention, the lipophilicmoicty is a lipophilic moiety as will be understood by those skilled inthe art. The lipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbonatoms. The lipophilic moiety preferably has between a lower limit of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Thelipophilic moiety more preferably has between a lower limit of 2, 3, 4,5, 6, 7, 8, 9, or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilicmoiety even more preferably has between a lower limit of 3, 4, 5, 6, 7;8, or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14carbon atoms. The lipophilic moiety still more preferably has between alower limit of 3, 4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10carbon atoms. The lipophilic moiety most preferably has 6 carbon atoms.The lipophilic moiety is preferably selected from the group consistingof saturated or unsaturated, linear or branched alkyl moieties,saturated or unsaturated, linear or branched fatty acid moieties,cholesterol, and adamantane. Exemplary alkyl moieties include, but arenot limited to, saturated, linear alkyl moieties such as methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,nonadecyl and eicosyl; saturated, branched alkyl moieties such asisopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; andunsaturated alkyl moieties derived from the above saturated alkylmoieties including, but not limited to, vinyl, allyl, 1-butenyl,2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Exemplary fatty acidmoieties include, but are not limited to, unsaturated fatty acidmoieties such as lauroleate, myristoleate, palmitoleate, oleate,elaidate, erucate, linoleate, linolenate, arachidonate,eicosapentaentoate, and docosahexaenoate; and saturated fatty acidmoieties such as acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate.

According to these embodiments of the present invention, the spacermoieties, G, G′ and G″, are spacer moieties as will be understood bythose skilled in the art. Spacer moieties are preferably selected fromthe group consisting of sugar moieties, cholesterol and glycerinemoieties. Sugar moieties may be various sugar moieties as will beunderstood by those skilled in the art including, but not limited to,monosaccharide moieties and disaccharide moieties. Preferredmonosaccharide moieties have between 4 and 6 carbon atoms. Preferably,oligomers of these embodiments do not include spacer moieties (i.e., k,m and n are preferably 0).

According to these embodiments of the present invention, the linkermoiety, L, may be used to couple the oligomer with the drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties. Thealkyl linker moiety may be a saturated or unsaturated, linear orbranched alkyl moiety as will be understood by those skilled in the artincluding, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Thealkoxy moiety may be various alkoxy moieties including, but not limitedto, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy,eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy,tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4,or 5 and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moietymay be a saturated or unsaturated, linear or branched fatty acid moietyas will be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

According to these embodiments of the present invention, the terminatingmoiety, T, is preferably an alkyl or alkoxy moiety. The alkyl or alkoxymoiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 carbon atoms. The alkyl or alkoxy moietymore preferably has between a lower limit of 1, 2, 3, 4, 5, 6, or 7 andan upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thealkyl or alkoxy moiety even more preferably has between a lower limit of1, 2, 3, 4, or 5 and an upper limit of 5, 6, 7, 8, 9, or 10 carbonatoms. The alkyl or alkoxy moiety still more preferably has between alower limit of 1, 2, 3, or 4 and an upper limit of 5, 6, or 7 carbonatoms. The alkyl moiety may be various linear or branched, saturated orunsaturated alkyl moieties as will be understood by those skilled in theart including, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary alkoxy moieties may be various alkoxy moieties including, butnot limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy,heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy,tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy,nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy,2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy,2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy,2-butenyloxy, ethynyloxy, 1-propynyloxy, and 2-propynyloxy. Theterminating moiety is more preferably a lower alkyl moiety such asmethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,or tert-pentyl, or a lower alkoxy moiety such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, ortert-pentyloxy. Most preferably, the terminating moiety is methyl ormethoxy. While the terminating moiety is preferably an alkyl or alkoxymoiety, it is to be understood that the terminating moiety may bevarious moieties as will be understood by those skilled in the artincluding, but not limited to, sugar moieties, cholesterol, alcohols,and fatty acid moieties.

According to these embodiments of the present invention, the activatablemoiety, A, is a moiety that allows for the coupling of the oligomer toan activating agent to form an activated oligomer capable of couplingwith the proinsulin polypeptide. The activatable moiety may be variousactivatable moieties as will be understood by those skilled in the artincluding, but not limited to, —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH,and NH₂.

In still other embodiments, the oligomer comprises the structure ofFormula II:A-X(CH₂)_(m)Y(C₂H₄O)_(n)R  (II)wherein:

A is —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH₂;

-   -   X is an oxygen atom or a covalent bond, with the proviso X is        not an oxygen atom when A is —OH;    -   Y is an ester, an ether, a carbamate, a carbonate, or an amide        bonding moiety, and is preferably an ether bonding moiety;    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In still other embodiments, the oligomer comprises the structure ofFormula III:A-(CH₂)_(m)(CH₂)_(m)(OC₂H₄)_(n)OR  (III)wherein:

-   -   A is —C(O)—OH, C(S)OH, —C(S)—SH, —OH, —SH, or NH₂;    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and

R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, analcohol moiety, or a fatty acid moiety. The alkyl moiety may be a linearor branched, saturated or unsaturated alkyl moieties as will beunderstood by those skilled in the art including, but not limited to,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, ten-butyl,2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl,ethynyl, 1-propynyl, and 2-propynyl. The alkyl moiety is more preferablya lower alkyl moiety such as methyl, ethyl, propyl, isopropyl, butyl,sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl moiety is stillmore preferably a C₁ to C₃ alkyl. The alkyl moiety is most preferablymethyl. The fatty acid moiety may be a saturated or unsaturated, linearor branched fatty acid moiety as will be understood by those skilled inthe art including, but not limited to, lauroleate, myristoleate,palmitoleate, oleate, elaidate, erucate, linoleate, linolenate,arachidonate, eicosapentaentoate, docosahexaenoate, acetate, caproate,caprylate, caprate, laurate, arachidate, behenate, lignocerate, andcerotate.

In yet other embodiments, the oligomer comprises the structure ofFormula IV:

wherein:

-   -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In still other embodiments, the oligomer comprises the structure ofFormula V:

In the various embodiments described above, the oligomer is covalentlycoupled to the insulin polypeptide. In some embodiments, the oligomer iscoupled to the insulin polypeptide utilizing a hydrolyzable bond (e.g.,an ester or carbonate bond). A hydrolyzable coupling may provide aninsulin polypeptide-oligomer conjugate that acts as a prodrug. Incertain instances, for example where the insulin polypeptide-oligomerconjugate is biologically inactive (i.e., the conjugate lacks theability to affect the body through the insulin polypeptide's primarymechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, providing the biologicallyactive insulin polypeptide over a given time period as one or moreoligomers are cleaved from their respective biologically inactiveinsulin polypeptide-oligomer conjugates to provide the biologicallyactive insulin polypeptide. In other embodiments, the oligomer iscoupled to the insulin polypeptide utilizing a non-hydrolyzable bond(e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzablebond may be preferable when it is desirable to allow the biologicallyinactive insulin polypeptide-oligomer conjugate to circulate in thebloodstream for an extended period of time, preferably at least 2 hours.When the oligomer is coupled to the insulin polypeptide utilizing abonding moiety that comprises a carbonyl moiety, such as an ester, acarbamate, a carbonate, or an amide bonding moiety, the resultinginsulin polypeptide-oligomer conjugate is an insulin polypeptide-acyloligomer conjugate.

Oligomers employed in the various embodiments described above arecommercially available or may be synthesized by various methods as willbe understood by those skilled in the art. For example, polydispersedoligomers may be synthesized by the methods provided in one or more ofthe following references: U.S. Pat. No. 4,179,337 to Davis et al.; U.S.Pat. No. 5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe;U.S. Pat. No., 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 toEkwuribe, U.S. Pat. No. 6,309,633 to Ekwuribe et al. Non-polydispersed(e.g., substantially monodispersed and monodispersed) oligomers may besynthesized by methods provided in one or more of the followingreferences: U.S. patent application Ser. No. 09/873,731 filed Jun. 4,2001 by Ekwuribe et al. entitled “Methods of Synthesizing SubstantiallyMonodispersed Mixtures of Polymers Having Polyethylene Glycol Mixtures”;U.S. patent application Ser. No. 09/873,797 filed Jun. 4, 2001 byEkwuribe et al. entitled “Mixtures of Drug-Oligomer ConjugatesComprising Polyalkylene Glycol, Uses Thereof, and Methods of MakingSame”; and U.S. patent application Ser. No. 09/873,899 filed Jun. 4,2001 by Ekwuribe et al. entitled “Mixtures of Insulin Drug-OligomerConjugates Comprising Polyalkylene Glycol, Uses Thereof, and Methods ofMaking Same”. Oligomers according to embodiments of the presentinvention are preferably substantially monodispersed and are morepreferably monodispersed. Exemplary methods for synthesizing preferredmonodispersed oligomers are provided in Examples 1 through 10 below.

The contacting of the proinsulin polypeptide with the oligomer underconditions sufficient to provide a proinsulin polypeptide-oligomerconjugate may be performed utilizing various conditions as will beunderstood by those skilled in the art. Preferably, the contacting ofthe proinsulin polypeptide with the oligomer under conditions sufficientto provide a proinsulin polypeptide-oligomer conjugate comprisescontacting the oligomer with an activating agent under conditionssufficient to provide an activated oligomer; and contacting theactivated oligomer with the proinsulin polypeptide under conditionssufficient to provide the proinsulin polypeptide conjugate. Theactivated oligomer may be formed ex situ or in situ.

The activating agent may be various activating agents capable ofactivating one or more of the oligomers described above so that theoligomer is capable of reacting with nucleophilic hydroxyl functionsand/or amino functions in the proinsulin polypeptide as will beunderstood by those skilled in the art including, but not limited to,N-hydroxysuccinimide, p-nitrophenyl chloroformate,1,3-dicyclohexylcarbodiimide, and hydroxybenzotriazide.

One skilled in the art will understand the conditions sufficient tocouple the activating agent to the oligomer to provide an activatedoligomer. For example, one skilled in the art can refer to R. C. Larock,COMPREHENSIVE ORGANIC TRANSFORMATIONS. A GUIDE TO FUNCTIONAL GROUPPREPARATIONS (2d Edition, N.Y., Wiley-VCH, 1999), the disclosure ofwhich is incorporated by reference herein in its entirety.

The conditions sufficient to couple the activated oligomer to theproinsulin polypeptide will be understood to one of skill in the art.For example, the proinsulin polypeptide may be dissolved in a dipolaraprotic solvent, such as dimethylsulfoxide, to provide a proinsulinpolypeptide solution. A buffering agent, such as triethylamine, may beadded to the proinsulin polypeptide solution. The activated oligomerdissolved in an anydrous solvent such as acetonitrile may then be addedto the proinsulin polypeptide solution. One skilled in the art may alsorefer to R. C. Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS. A GUIDE TOFUNCTIONAL GROUP PREPARATIONS (2d Edition, New York, Wiley-VCH, 1999).The molar ratio of activated oligomer to proinsulin polypeptide ispreferably greater than about 1:1, is more preferably greater than about2:1, is even more preferably greater than about 3:1, is still morepreferably greater than about 4:1, and is still even more preferablygreater than about 5:1.

In the various embodiments described above, more than one oligomer(i.e., a plurality of oligomers) may be coupled to the insulinpolypeptide portion of the proinsulin polypeptide. The oligomers in theplurality are preferably the same. However, it is to be understood thatthe oligomers in the plurality may be different from one another, or,alternatively, some of the oligomers in the plurality may be the sameand some may be different. When a plurality of oligomers are coupled tothe insulin polypeptide portion of the proinsulin polypeptide, it may bepreferable to couple one or more of the oligomers to the insulinpolypeptide portion of the proinsulin polypeptide with hydrolyzablebonds and couple one or more of the oligomers to the insulin polypeptideportion of the proinsulin polypeptide with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin polypeptide portion of the proinsulin polypeptide may behydrolyzable, but have varying degrees of hydrolyzability such that, forexample, one or more of the oligomers is rapidly removed from theinsulin polypeptide or insulin polypeptide portion of the proinsulinpolypeptide by hydrolysis in the body and one or more of the oligomersis slowly removed from the insulin polypeptide or insulin polypeptideportion by hydrolysis in the body.

In the various embodiments described above, the oligomer may be coupledto the insulin polypeptide portion of the proinsulin polypeptide atvarious nucleophilic residues of the insulin polypeptide portionincluding, but not limited to, nucleophilic hydroxyl functions and/oramino functions. A nucleophilic hydroxyl function may be found, forexample, at serine and/or tyrosine residues, and a nucleophilic aminofunction may be found, for example, at histidine and/or lysine residues,and/or at the one or more N-termini of the polypeptide. When an oligomeris coupled to the one or more N-termini of the proinsulin polypeptide,the coupling preferably forms a secondary amine. When the proinsulinpolypeptide has a leader peptide coupled to the N-terminus of theB-chain polypeptide, the N-termini of the insulin molecule may beprotected from conjugation (e.g., acylation). When the proinsulinpolypeptide is human proinsulin having a leader peptide coupled to theN-terminus of the B-chain, for example, the oligomer may be coupled tothe three amino functionalities of the proinsulin: the N-terminus of theleader peptide, the amino functionality of the Lys residue in theC-peptide, and the amino functionality of LysB²⁹. Upon cleavage of theleader peptide and the C-peptide, one finds that the oligomer has beensite specifically coupled to the Lys^(B29) of the insulin to provide asingle insulin conjugate, insulin mono-conjugated with an oligomer atLys^(B29).

The cleaving of the one or more peptides from the proinsulinpolypeptide-oligomer conjugate to provide the insulinpolypeptide-oligomer conjugate may be performed by various processes aswill be understood by those skilled in the art. Preferably, the cleavingof the one or more peptides from the proinsulin polypeptide-oligomerconjugate comprises contacting the proinsulin polypeptide-oligomerconjugate with one or more enzymes that are capable of cleaving thebond(s) between the one or more peptides and the insulin polypeptideunder conditions sufficient to cleave the one or more peptides from theproinsulin polypeptide-oligomer conjugate. As described in variousreferences, for example, Kemmler et al. “Studies on the Conversion ofProinsulin to Insulin,” J. Biol. Chem., 246: 6786-6791 (1971), thedisclosure of which is incorporated herein by reference in its entirety,one skilled in the art will understand how to select appropriate enzymesin view of the particular peptide bond(s) to be cleaved and how toprovide conditions sufficient to cleave the one or more peptides fromthe proinsulin polypeptide-oligomer conjugate. The one or more enzymespreferably comprise various enzymes including, but not limited to,trypsin, chymotrypsin, carboxy peptidase B, and mixtures thereof. Morepreferably, the one or more enzymes are selected from the groupconsisting of trypsin, carboxy peptidase B, and mixtures thereof.

In some embodiments such as those described above having a connectingpeptide, the connecting peptide has a terminal amino acid residue at thefirst end. In some of these embodiments, the cleaving of the connectingpeptide from the proinsulin polypeptide-oligomer conjugate comprisescontacting the proinsulin polypeptide-oligomer conjugate with a firstenzyme under conditions sufficient to provide a terminal amino acidresidue-insulin polypeptide-oligomer conjugate, and contacting theterminal amino acid residue-insulin polypeptide-oligomer conjugate witha second enzyme under conditions sufficient to provide the insulinpolypeptide-oligomer conjugate. The contacting of the proinsulinpolypeptide-oligomer conjugate with a first enzyme and the contacting ofthe terminal amino acid residue-insulin polypeptide-oligomer conjugatewith a second enzyme may occur substantially concurrently, for examplewhen the first enzyme and the second enzyme are provided as a mixture orcocktail. Preferably, the first enzyme is trypsin and the second enzymeis carboxy peptidase B. The terminal amino acid residue may be variousresidues, such as an arginine residue. For example, the terminal aminoacid residue is an arginine residue when the insulin polypeptide isinsulin and the connecting peptide is human C-peptide.

The cleaving of the one or more peptides from the proinsulinpolypeptide-oligomer conjugate preferably provides an insulinpolypeptide-oligomer conjugate product that consists of a single insulinpolypeptide-oligomer conjugate (i.e., is substantially devoid ofadditional insulin polypeptide-oligomer conjugates). Preferably, theinsulin polypeptide-oligomer conjugate product consists of a singleinsulin polypeptide-oligomer monoconjugate. For example, in embodimentsdescribed above in which the proinsulin polypeptide comprises an insulinpolypeptide having an A-chain polypeptide devoid of lysine residues anda B-chain polypeptide comprising a single lysine residue, the insulinpolypeptide-oligomer conjugate product preferably consists of a singleinsulin polypeptide-oligomer monoconjugate where the oligomer is coupledto the lysine residue of the B-chain polypeptide. As another example,when the proinsulin polypeptide is proinsulin with a leader peptide, thecleaving of the C-peptide and the leader peptide from theproinsulin-oligomer conjugate provides an insulin-oligomermonoconjugate, wherein the insulin is monoconjugated at Lys^(B29).

The embodiments of the methods for synthesizing insulinpolypeptide-oligomer conjugates described above preferably result in ayield of insulin polypeptide-oligomer conjugates that is greater than75, 76, 77, 78, or 79 percent. More preferably, the yield is greaterthan 80, 81, 82, 83, 84, or 85 percent. Even more preferably, the yieldis greater than 86, 87, 88, 89, or 90 percent. Still more preferably,the yield is greater than 91, 92, 93, 94, or 95 percent. When theproinsulin polypeptide-oligomer conjugate is provided by contacting anactivated oligomer with the proinsulin polypeptide-oligomer conjugate,it may be preferable to use an excess of activated oligomers inachieving higher yields. For example, yields described above arepreferably obtained by using a molar ratio of activated oligomer toproinsulin polypeptide of greater than about 2:1, more preferablygreater than about 3:1, even more preferably greater than about 4:1, andstill more preferably greater than about 5:1. Preferably, yields greaterthan 91, 92, 93, 94, or 95 percent are obtained using a molar ratio ofactivated oligomer to proinsulin polypeptide of greater than about 4:1,more preferably greater than about 5:1.

According to other embodiments of the present invention, methods ofsynthesizing a proinsulin polypeptide-oligomer conjugate includecontacting a proinsulin polypeptide comprising an insulin polypeptidecoupled to one or more peptides by peptide bond(s) capable of beingcleaved to yield the insulin polypeptide with an oligomer underconditions sufficient to couple the oligomer to the insulin polypeptideportion of the proinsulin polypeptide and provide a proinsulinpolypeptide-oligomer conjugate. For example, proinsulin-oligomerconjugates may be synthesized as described in the Examples providedbelow. An embodiment of a synthesis route is provided in FIG. 1.

The proinsulin polypeptide may be various proinsulin polypeptidescomprising an insulin polypeptide coupled to one or more peptides bypeptide bond(s) capable of being cleaved to yield the insulinpolypeptide as will be understood by those skilled in the art including,but not limited to, proinsulin, proinsulin analogs, proinsulinfragments, proinsulin fragment analogs, miniproinsulin, or fusionproteins. In some embodiments, the proinsulin polypeptide is aproinsulin analog having a leader peptide. The proinsulin analog havinga leader peptide is produced by Itoham Foods, Inc. of Ibaraki Pref,Japan. The leader peptide and the C-peptide of the proinsulin analog areeach devoid of lysine residues. In other embodiments, the proinsulinpolypeptide is a proinsulin poplypeptide produced by Biobras of BeloHorizonte, Brazil. The proinsulin polypeptide has a leader peptidecoupled to the N-terminus of the B-chain of the proinsulin. The leaderpeptide is devoid of lysine residues.

The insulin polypeptide preferably has an A-chain polypeptide and aB-chain polypeptide. The A-chain polypeptide is preferably devoid oflysine residues. The B-chain polypeptide preferably comprises a singlelysine residue. The A-chain polypeptide and the B-chain polypeptide arepreferably cross-linked, and are more preferably cross-linked using oneor more disulfide bonds. Still more preferably, the A-chain polypeptideand the B-chain polypeptide each comprise cysteine residues, one or moreof which are coupled using one or more disulfide bonds to cross-link theA-chain polypeptide with the B-chain polypeptide. Preferably, theinsulin polypeptide is insulin, an insulin analog, an insulin fragment,or an insulin analog fragment.

In some embodiments, the one or more peptides coupled to the insulinpolypeptide comprise a connecting peptide coupled at a first end to theC-terminus of the B-chain polypeptide and at a second end to theN-terminus of the A-chain polypeptide. In general, the amino acidsequence of the connecting peptide is not critical and the connectingpeptide may be various connecting peptides as will be understood bythose skilled in the art including, but not limited to, C-peptidepolypeptides, C-peptides, and the connecting peptides inminiproinsulins. In some embodiments, the connecting peptide is devoidof lysine residues.

These embodiments may utilize less oligomeric reagents by reducing thenumber of possible conjugation sites on the proinsulin polypeptidemolecule.

In other embodiments, the one or more peptides coupled to the insulinpolypeptide comprise a leader peptide that is coupled to the N-terminusof the B-chain polypeptide. In general, the amino acid sequence of theleader peptide is not critical. In some embodiments, the leader peptideis devoid of lysine residues. These embodiments may reduce the amount ofoligomeric reagent used by limiting the number of conjugation sites onthe proinsulin polypeptide molecule.

In still other embodiments, the one or more peptides coupled to theinsulin polypeptide comprise both a connecting peptide as describedabove and a leader peptide as described above. The one or more peptidesmay consist essentially of a connecting peptide and a leader peptide, ormay consist of a connecting peptide and a leader peptide.

The peptide bonds are bonds that may be cleaved in various ways as willbe understood by those skilled in the art. Preferably, the peptide bondsare bonds that may be enzymatically cleaved by enzymes including, butnot limited to, trypsin, carboxy peptidase B, thrombin, pepsin, andchymotripsin. Peptide bonds that may be enzymatically cleaved will beunderstood by those skilled in the art and include, but are not limitedto, Arg-Arg, Thr-Arg, Ala-Arg, Thr-Arg-Arg, Thr-Lys, Arg-Gly, andArg-Phe.

The oligomer may be various oligomers as will be understood by thoseskilled in the art. In general, the oligomer may be any oligomer capableof being coupled to a polypeptide as will be understood by those skilledin the art. For example, the oligomer may be a poly-dispersed oligomeras described in U.S. Pat. No. 4,179,337 to Davis et al.; U.S. Pat. No.5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe; U.S. Pat.No. 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 to Ekwuribe, and U.S.Pat. No. 6,309,633 to Ekwuribe et al. As another example, the oligomermay be a non-polydispersed oligomer as C, described in U.S. patentapplication Ser. No. 09/873,731 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Methods of Synthesizing Substantially Monodispersed Mixturesof Polymers Having Polyethylene Glycol Mixtures”; U.S. patentapplication Ser. No. 09/873,797 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Mixtures of Drug-Oligomer Conjugates Comprising PolyalkyleneGlycol, Uses Thereof, and Methods of Making Same”; and U.S. patentapplication Ser. No. 09/873,899 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Mixtures of Insulin Drug-Oligomer Conjugates ComprisingPolyalkylene Glycol, Uses Thereof, and Methods of Making Same.”

The oligomer preferably comprises a hydrophilic moiety as will beunderstood by those skilled in the art including, but not limited to,polyalkylene glycols such as polyethylene glycol or polypropyleneglycol, polyoxyethylenated polyols, copolymers thereof and blockcopolymers thereof provided that the hydrophilicity of the blockcopolymers is maintained. The hydrophilic moiety is preferably apolyalkylene glycol moiety. The polyalkylene glycol moiety has at least1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. Thepolyalkylene glycol moiety more preferably has between a lower limit of2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 polyalkylene glycol subunits. Even morepreferably, the polyalkylene glycol moiety has between a lower limit of3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, or 12polyalkylene glycol subunits. The polyalkylene glycol moiety still morepreferably has between a lower limit of 4, 5, or 6 and an upper limit of6, 7, or 8 polyalkylene glycol subunits. The polyalkylene glycol moietymost preferably has 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety of the oligomer is preferably a lower alkyl polyalkyleneglycol moiety such as a polyethylene glycol moiety, a polypropyleneglycol moiety, or a polybutylene glycol moiety. When the polyalkyleneglycol moiety is a polypropylene glycol moiety, the moiety preferablyhas a uniform (i.e., not random) structure. An exemplary polypropyleneglycol moiety having a uniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

The oligomer may further comprise one or more additional hydrophilicmoieties (i.e., moieties in addition to the polyalkylene glycol moiety)including, but not limited to, sugars, polyalkylene glycols, andpolyamine(PEG copolymers. Adjacent polyalkylene glycol moieties will beconsidered to be the same moiety if they are coupled by ether bonds. Forexample, the moiety—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄— —C₂H₄— —C₂H₄—is a single polyethylene glycol moiety having six polyethylene glycolsubunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

is a polyethylene glycol moiety having four polyethylene glycol subunitsand an additional hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and no additionalhydrophilic moieties.

The oligomer preferably further comprises one or more lipophilicmoieties as will be understood by those skilled in the art. Thelipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbon atoms. Thelipophilic moiety preferably has between a lower limit of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. The lipophilicmoiety more preferably has between a lower limit of 2, 3, 4, 5, 6, 7, 8,9, or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilic moiety even morepreferably has between a lower limit of 3, 4, 5, 6, 7, 8, or 9 and anupper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thelipophilic moiety still more preferably has between a lower limit of 3,4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10 carbon atoms. Thelipophilic moiety most preferably has 6 carbon atoms. The lipophilicmoiety is preferably selected from the group consisting of saturated orunsaturated, linear or branched alkyl moieties, saturated orunsaturated, linear or branched fatty acid moieties, cholesterol, andadamantane. Exemplary alkyl moieties include, but are not limited to,saturated, linear alkyl moieties such as methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl;saturated, branched alkyl moieties such as isopropyl, sec-butyl,tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derivedfrom the above saturated alkyl moieties including, but not limited to,vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary fatty acid moieties include, but are not limited to,unsaturated fatty acid moieties such as lauroleate, myristoleate,palmitoleate, oleate, elaidate, erucate, linoleate, linolenate,arachidonate, eicosapentaentoate, and docosahexaenoate; and saturatedfatty acid moieties such as acetate, caproate, caprylate, caprate,laurate, arachidate, behenate, lignocerate, and cerotate.

The oligomer may further comprise one or more spacer moieties as will beunderstood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theproinsulin polypeptide, to separate a first hydrophilic or lipophilicmoiety from a second hydrophilic or lipophilic moiety, or to separate ahydrophilic moiety or lipophilic moiety from a linker moiety. Spacermoieties are preferably selected from the group consisting of sugar,cholesterol and glycerine moieties. Sugar moieties may be various sugarmoieties as will be understood by those skilled in the art including,but not limited to, monosaccharide moieties and disaccharide moieties.Preferred monosaccharide moieties have between 4 and 6 carbon atoms.

The oligomer may further comprise one or more linker moieties that areused to couple the oligomer with the proinsulin polypeptide as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties. Theallyl linker moiety may be a saturated or unsaturated, linear orbranched alkyl moiety as will be understood by those skilled in the artincluding, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Thealkoxy moiety may be various alkoxy moieties including, but not limitedto, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy,eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy,tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4, or 5and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moiety maybe a saturated or unsaturated, linear or branched fatty acid moiety aswill be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

The oligomer may further comprise one or more terminating moieties atthe one or more ends of the oligomer, which are not coupled to theinsulin polypeptide. The terminating moiety is preferably an alkyl oralkoxy moiety. The alkyl or alkoxy moiety preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbonatoms. The alkyl or alkoxy moiety more preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, or 7 and an upper limit of 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 carbon atoms. The alkyl or alkoxy moiety even morepreferably has between a lower limit of 1, 2, 3, 4, or 5 and an upperlimit of 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl or alkoxy moietystill more preferably has between a lower limit of 1, 2, 3, or 4 and anupper limit of 5, 6, or 7 carbon atoms. The alkyl moiety may be a linearor branched, saturated or unsaturated alkyl moiety as will be understoodby those skilled in the art including, but not limited to, methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl,2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl,ethynyl, 1-propynyl, and 2-propynyl. The alkoxy moiety may be variousalkoxy moieties including, but not limited to, methoxy, ethoxy, propoxy,butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy,sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy,2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The terminating moiety ismore preferably a lower alkyl moiety such as methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl, or alower alkoxy moiety such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. Mostpreferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

According to other embodiments of the present invention, the oligomercomprises the structure of Formula VI:A-L_(j)-G_(k)-R-G′_(m)-R′-G′_(n)-T  (VI)wherein:

-   -   A is an activatable moiety,    -   L is a linker moiety;    -   G, G′ and G″ are individually selected spacer moieties;    -   R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,        or R′ is the lipophilic moiety and R is the polyalkylene glycol        moiety,    -   T is a terminating moiety; and    -   j, k, m and n are individually 0 or 1.

According to these embodiments of the present invention, thepolyalkylene glycol moiety has at least 1, 2, 3, 4, 5, 6 or 7polyalkylene glycol subunits. The polyalkylene glycol moiety preferablyhas between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more polyalkylene glycol subunits. The polyalkylene glycol moietymore preferably has between a lower limit of 2, 3, 4, 5, or 6 and anupper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 polyalkylene glycol subunits. Even more preferably, the polyalkyleneglycol moiety has between a lower limit of 3, 4, 5, or 6 and an upperlimit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol subunits. Thepolyalkylene glycol moiety still more preferably has between a lowerlimit of 4, 5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycolsubunits. The polyalkylene glycol moiety most preferably has 7polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene glycol moiety is apolypropylene glycol moiety, the moiety preferably has a uniform (i.e.,not random) structure. An exemplary polypropylene glycol moiety having auniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

According to these embodiments of the present invention, the lipophilicmoiety is a lipophilic moiety as will be understood by those skilled inthe art. The lipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbonatoms. The lipophilic moiety preferably has between a lower limit of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Thelipophilic moiety more preferably has between a lower limit of 2, 3, 4,5, 6, 7, 8, 9, or 10-and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilicmoiety even more preferably has between a lower limit of 3, 4, 5, 6, 7,8, or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14carbon atoms. The lipophilic moiety still more preferably has between alower limit of 3, 4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10carbon atoms. The lipophilic moiety most preferably has 6 carbon atoms.The lipophilic moiety is preferably selected from the group consistingof saturated or unsaturated, linear or branched alkyl moieties,saturated or unsaturated, linear or branched fatty acid moieties,cholesterol, and adamantane. Exemplary alkyl moieties include, but arenot limited to, saturated, linear alkyl moieties such as methyl, ethyl,propyl, butyl pentyl, hexyl, heptyl, octyl, nonyl, decyl undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,nonadecyl and eicosyl; saturated, branched alkyl moieties such asisopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; andunsaturated alkyl moieties derived from the above saturated alkylmoieties including, but not limited to, vinyl, allyl, 1-butenyl,2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Exemplary fatty acidmoieties include, but are not limited to, unsaturated fatty acidmoieties such as lauroleate, myristoleate, palmitoleate, oleate,elaidate, erucate, linoleate, linolenate, arachidonate,eicosapentaentoate, and docosahexaenoate; and saturated fatty acidmoieties such as acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate.

According to these embodiments of the present invention, the spacermoieties, G, G′ and G″, are spacer moieties as will be understood bythose skilled in the art. Spacer moieties are preferably selected fromthe group consisting of sugar moieties, cholesterol and glycerinemoieties. Sugar moieties may be various sugar moieties as will beunderstood by those skilled in the art including, but not limited to,monosaccharide moieties and disaccharide moieties. Preferredmonosaccharide moieties have between 4 and 6 carbon atoms. Preferably,oligomers of these embodiments do not include spacer moieties (i.e., k,m and n are preferably 0).

According to these embodiments of the present invention, the linkermoiety, L, may be used to couple the oligomer with the drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties. Thealkyl linker moiety may be a saturated or unsaturated, linear orbranched alkyl moiety as will be understood by those skilled in the artincluding, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl; dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Thealkoxy moiety may be various alkoxy moieties including, but not limitedto, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy,eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy,tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4, or 5and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moiety maybe a saturated or unsaturated, linear or branched fatty acid moiety aswill be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

According to these embodiments of the present invention, the terminatingmoiety, T, is preferably an alkyl or alkoxy moiety. The alkyl or alkoxymoiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 carbon atoms. The alkyl or alkoxy moietymore preferably has between a lower limit of 1, 2, 3, 4, 5, 6, or 7 andan upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thealkyl or alkoxy moiety even more preferably has between a lower limit of1, 2, 3, 4, or 5 and an upper limit of 5, 6, 7, 8, 9, or 10 carbonatoms. The alkyl or alkoxy moiety still more preferably has between alower limit of 1, 2, 3, or 4 and an upper limit of 5, 6, or 7 carbonatoms. The alkyl moiety may be various linear or branched, saturated orunsaturated alkyl moieties as will be understood by those skilled in theart including, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary alkoxy moieties may be various alkoxy moieties including, butnot limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy,heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy,tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy,nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy,2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy,2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy,2-butenyloxy, ethynyloxy, 1-propynyloxy, and 2-propynyloxy. Theterminating moiety is more preferably a lower alkyl moiety such asmethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,or tert-pentyl, or a lower alkoxy moiety such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, ortert-pentyloxy. Most preferably, the terminating moiety is methyl ormethoxy. While the terminating moiety is preferably an alkyl or alkoxymoiety, it is to be understood that the terminating moiety may bevarious moieties as will be understood by those skilled in the artincluding, but not limited to, sugar moieties, cholesterol, alcohols,and fatty acid moieties.

According to these embodiments of the present invention, the activatablemoiety, A, is a moiety that allows for the coupling of the oligomer toan activating agent to form an activated oligomer capable of couplingwith the proinsulin polypeptide. The activatable moiety may be variousactivatable moieties as will be understood by those skilled in the artincluding, but not limited to, —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH,and NH₂.

In still other embodiments, the oligomer comprises the structure ofFormula VII:A-X(CH₂)_(m)Y(C₂H₄O)_(n)R  (VII)wherein:

-   -   A is —C(O)—OH, C(S)OH, —C(S)SH, —OH, —SH, or NH₂;    -   X is an oxygen atom or a covalent bond, with the proviso X is        not an oxygen atom when A is —OH;    -   Y is an ester, an ether, a carbamate, a carbonate, or an amide        bonding moiety, and is preferably an ether bonding moiety;    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In still other embodiments, the oligomer comprises the structure ofFormula VIII:A—(CH₂)_(m)(OC₂H₄)_(n)OR  (VIII)wherein:

-   -   A is —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH₂;    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In yet other embodiments, the oligomer comprises the structure ofFormula IX:

wherein:

-   -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl        butyl sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In still other embodiments, the oligomer comprises the structure ofFormula X:

In the various embodiments of methods for synthesizing proinsulinpolypeptide-oligomer conjugates described above, the oligomer iscovalently coupled to the insulin polypeptide. In some embodiments, theoligomer is coupled to the insulin polypeptide utilizing a hydrolyzablebond (e.g., an ester or carbonate bond). A hydrolyzable coupling mayprovide an insulin polypeptide-oligomer conjugate that acts as aprodrug. In certain instances, for example where the insulinpolypeptide-oligomer conjugate is biologically inactive (i.e., theconjugate lacks the ability to affect the body through the insulinpolypeptide's primary mechanism of action), a hydrolyzable coupling mayprovide for a time-release or controlled-release effect, providing thebiologically active insulin polypeptide over a given time period as oneor more oligomers are cleaved from their respective biologicallyinactive insulin polypeptide-oligomer conjugates to provide thebiologically active insulin polypeptide. In other embodiments, theoligomer is coupled to the insulin polypeptide utilizing anon-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use ofa non-hydrolyzable bond may be preferable when it is desirable to allowthe biologically inactive insulin polypeptide-oligomer conjugate tocirculate in the bloodstream for an extended period of time, preferablyat least 2 hours. When the oligomer is coupled to the insulinpolypeptide utilizing a bonding moiety that comprises a carbonyl moiety,such as an ester, a carbamate, a carbonate, or an amide bonding moiety,the resulting insulin polypeptide-oligomer conjugate is an insulinpolypeptide-acyl oligomer conjugate.

Oligomers employed in the embodiments of methods for synthesizingproinsulin polypeptide oligomer conjugates described above arecommercially available or may be synthesized by various methods as willbe understood by those skilled in the art. For example, polydispersedoligomers may be synthesized by the methods provided in one or more ofthe following references: U.S. Pat. No. 4,179,337 to Davis et al.; U.S.Pat. No. 5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe;U.S. Pat. No. 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 toEkwuribe, U.S. Pat. No. 6,309,633 to Ekwuribe et al. Non-polydispersed(e.g., substantially monodispersed and monodispersed) oligomers may besynthesized by methods provided in one or more of the followingreferences: U.S. patent application Ser. No. 09/873,731 filed Jun. 4,2001 by Ekwuribe et al. entitled “Methods of Synthesizing SubstantiallyMonodispersed Mixtures of Polymers Having Polyethylene Glycol Mixtures”;U.S. patent application Ser. No. 09/873,797 filed Jun. 4, 2001 byEkwuribe et al. entitled “Mixtures of Drug-Oligomer ConjugatesComprising Polyalkylene Glycol, Uses Thereof, and Methods of MakingSame”; and U.S. patent application Ser. No. 09/873,899 filed Jun. 4,2001 by Ekwuribe et al. entitled “Mixtures of Insulin Drug-OligomerConjugates Comprising Polyalkylene Glycol, Uses Thereof, and Methods ofMaking Same”. Oligomers according to embodiments of the presentinvention are preferably substantially monodispersed and are morepreferably monodispersed. Exemplary methods for synthesizing preferredmonodispersed oligomers are provided in Examples 1 through 10 below.

The contacting of the proinsulin polypeptide with the oligomer underconditions sufficient to provide a proinsulin polypeptide-oligomerconjugate may be performed utilizing various conditions as will beunderstood by those skilled in the art. Preferably, the contacting ofthe proinsulin polypeptide with the oligomer under conditions sufficientto provide a proinsulin polypeptide-oligomer conjugate comprisescontacting the oligomer with an activating agent under conditionssufficient to provide an activated oligomer; and contacting theactivated oligomer with the proinsulin polypeptide under conditionssufficient to provide the proinsulin polypeptide conjugate. Theactivated oligomer may be formed ex situ or in situ.

The activating agent may be various activating agents capable ofactivating one or more of the oligomers described above so that theoligomer is capable of reacting with nucleophilic hydroxyl functionsand/or amino functions in the proinsulin polypeptide as will beunderstood by those skilled in the art including, but not limited to,N-hydroxysuccinimide, p-nitrophenyl chloroformate,1,3-dicyclohexylcarbodiimide, and hydroxybenzotriazide.

One skilled in the art will understand the conditions sufficient tocouple the activating agent to the oligomer to provide an activatedoligomer. For example, one skilled in the art can refer to R. C. Larock,COMPREHENSIVE ORGANIC TRANSFORMATIONS. A GUIDE TO FUNCTIONAL GROUPPREPARATONS (2d Edition, N.Y., Wiley-VCH, 1999).

The conditions sufficient to couple the activated oligomer to theproinsulin polypeptide will be understood to one of skill in the art.For example, the proinsulin polypeptide may be dissolved in a dipolaraprotic solvent, such as dimethylsulfoxide, to provide a proinsulinpolypeptide solution. A buffering agent, such as triethylamine, may beadded to the proinsulin polypeptide solution. The activated oligomerdissolved in an anydrous solvent such as acetonitrile may then be addedto the proinsulin polypeptide solution. One skilled in the art may alsorefer to R. C. Larock, COMPREHENSrVE ORGANIC TRANSFORMATIONS. A GUIDE TOFUNCTIONAL GROUP PREPARATIONS (2d Edition, N.Y., Wiley-VCH, 1999). Themolar ratio of activated oligomer to proinsulin polypeptide ispreferably greater than about 1:1, is more preferably greater than about2:1, is even more preferably greater than about 3:1, is still morepreferably greater than about 4:1, and is still even more preferablygreater than about 5:1.

In the various embodiments of methods for synthesizing proinsulinpolypeptide-oligomer conjugates described above, more than one oligomer(i.e., a plurality of oligomers) may be coupled to the insulinpolypeptide portion of the proinsulin polypeptide. The oligomers in theplurality are preferably the same. However, it is to be understood thatthe oligomers in the plurality may be different from one another, or,alternatively, some of the oligomers in the plurality may be the sameand some may be different. When a plurality of oligomers are coupled tothe insulin polypeptide portion of the proinsulin polypeptide, it may bepreferable to couple one or more of the oligomers to the insulinpolypeptide portion of the proinsulin polypeptide with hydrolyzablebonds and couple one or more of the oligomers to the insulin polypeptideportion of the proinsulin polypeptide with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin polypeptide portion of the proinsulin polypeptide may behydrolyzable, but have varying degrees of hydrolyzability such that, forexample, one or more of the oligomers is rapidly removed from theinsulin polypeptide or insulin polypeptide portion of the proinsulinpolypeptide by hydrolysis in the body and one or more of the oligomersis slowly removed from the insulin polypeptide or insulin polypeptideportion by hydrolysis in the body.

In the various embodiments of methods for synthesizing proinsulinpolypeptide-oligomer conjugates described above, the oligomer may becoupled to the insulin polypeptide portion of the proinsulin polypeptideat various nucleophilic residues of the insulin polypeptide portionincluding, but not limited to, nucleophilic hydroxyl functions and/oramino functions. A nucleophilic hydroxyl function may be found, forexample, at serine and/or tyrosine residues, and a nucleophilic aminofunction may be found, for example, at histidine and/or lysine residues,and/or at the one or more N-termini of the polypeptide. When an oligomeris coupled to the one or more N-termini of the proinsulin polypeptide,the coupling preferably forms a secondary amine. When the proinsulinpolypeptide has a leader peptide coupled to the N-terminus of theB-chain polypeptide, the N-termini of the insulin molecule may beprotected from conjugation (e.g., acylation). When the proinsulinpolypeptide is human proinsulin having a leader peptide coupled to theN-terminus of the B-chain, for example, the oligomer may be coupled tothe three amino functionalities of the proinsulin: the N-terminus of theleader peptide, the amino functionality of the Lys residue in theC-peptide, and the amino functionality of LYS^(B29). Upon cleavage ofthe leader peptide and the C-peptide, one finds that the oligomer hasbeen site specifically coupled to the Lys^(B29) of the insulin toprovide a single insulin conjugate, insulin mono-conjugated with anoligomer at Lys^(B29).

According to still other embodiments of the present invention, aproinsulin polypeptide-oligomer conjugate includes a proinsulinpolypeptide comprising an insulin polypeptide coupled to one or morepeptides by peptide bond(s) that are cleavable to yield the insulinpolypeptide, and an oligomer coupled to the insulin polypeptide portionof the proinsulin polypeptide.

The proinsulin polypeptide comprising an insulin polypeptide coupled toone or more peptides by peptide bond(s) that are cleavable to yield theinsulin polypeptide may be various proinsulin polypeptides including,but not limited to, the proinsulin polypeptides described above withreference to the methods of synthesizing proinsulin polypeptide-oligomerconjugates. The oligomer may be various oligomers including, but notlimited to, the oligomers described above with reference to the methodsof synthesizing proinsulin polypeptide-oligomer conjugates. The oligomerpreferably comprises a hydrophilic moiety and a lipophilic moiety.Proinsulin polypeptide-oligomer conjugates according to the presentinvention may be synthesized by various methods as will be understood bythose skilled in the art including, but not limited to, the methods ofsynthesizing proinsulin polypeptide-oligomer conjugates described above.

According to yet other embodiments, a method of synthesizing a C-peptidepolypeptide-oligomer conjugate includes contacting a pro-C-peptidepolypeptide comprising a C-peptide polypeptide coupled to one or morepeptides by peptide bond(s) that are cleavable to yield the C-peptidepolypeptide with an oligomer under conditions sufficient to couple theoligomer to the C-peptide polypeptide portion of the pro-C-peptidepolypeptide and provide a pro-C-peptide polypeptide-oligomer conjugate,and cleaving the one or more peptides from the pro-C-peptidepolypeptide-oligomer conjugate to provide the C-peptidepolypeptide-oligomer conjugate.

The pro-C-peptide polypeptide may be various pro-C-peptide polypeptidesas will be understood by those skilled in the art. Preferably, thepro-C-peptide polypeptide is a proinsulin polypeptide, and, morepreferably, the pro-C-peptide polypeptide is proinsulin.

The C-peptide polypeptide may be various C-peptide polypeptides as willbe understood by those skilled in the art. Preferably, the C-peptidepolypeptide is C-peptide.

The one or more peptides coupled to the C-peptide polypeptide may bevarious peptides as will be understood by those skilled in the art.Preferably, the one or more peptides comprise an insulin polypeptide.More preferably, the one or more polypeptides is an insulin polypeptide.The insulin polypeptide may be devoid of lysine residues, which mayreduce the amount of oligomeric reagents utilized to conjugate thepro-C-peptide polypeptide. Still more preferably, the one or morepeptides is insulin or insulin coupled at the N-terminus of the B-chainto a leader peptide.

The peptide bonds are bonds that may be cleaved in various ways as willbe understood by those skilled in the art. Preferably, the peptide bondsare bonds that may be enzymatically cleaved by enzymes including, butnot limited to, trypsin, carboxy peptidase B, thrombin, pepsin, andchymotripsin. Peptide bonds that may be enzymatically cleaved will beunderstood by those skilled in the art and include, but are not limitedto, Arg-Arg, Thr-Arg, Ala-Arg, Thr-Arg-Arg, Thr-Lys, Arg-Gly, andArg-Phe.

The oligomer may be various oligomers as will be understood by thoseskilled in the art. In general, the oligomer may be any oligomer capableof being coupled to a polypeptide as will be understood by those skilledin the art. For example, the oligomer may be a poly-dispersed oligomeras described in U.S. Pat. No. 4,179,337 to Davis et al.; U.S. Pat. No.5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe; U.S. Pat.No. 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 to Ekwuribe, and U.S.Pat. No. 6,309,633 to Ekwuribe et al. As another example, the oligomermay be a non-polydispersed oligomer as described in U.S. patentapplication Ser. No. 09/873,731 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Methods of Synthesizing Substantially Monodispersed Mixturesof Polymers Having Polyethylene Glycol Mixtures”; U.S. patentapplication Ser. No. 09/873,797 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Mixtures of Drug-Oligomer Conjugates Comprising PolyalkyleneGlycol, Uses Thereof, and Methods of Making Same”; and U.S. patentapplication Ser. No. 09/873,899 filed Jun. 4, 2001 by Ekwuribe et al.entitled “Mixtures of Insulin Drug-Oligomer Conjugates ComprisingPolyalkylene Glycol, Uses Thereof, and Methods of Making Same.”

The oligomer preferably comprises a hydrophilic moiety as will beunderstood by those skilled in the art including, but not limited to,polyalkylene glycols such as polyethylene glycol or polypropyleneglycol, polyoxyethylenated polyols, copolymers thereof and blockcopolymers thereof, provided that the hydrophilicity of the blockcopolymers is maintained. The hydrophilic moiety is preferably apolyalkylene glycol moiety. The polyalkylene glycol moiety has at least1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. Thepolyalkylene glycol moiety more preferably has between a lower limit of2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 polyalkylene glycol subunits. Even morepreferably, the polyalkylene glycol moiety has between a lower limit of3, 4, 5, or 6 and an upper limit of 5, 6, 7, 8, 9, 10, 11, or 12polyalkylene glycol subunits. The polyalkylene glycol moiety still morepreferably has between a lower limit of 4, 5, or 6 and an upper limit of6, 7, or 8 polyalkylene glycol subunits. The polyalkylene glycol moietymost preferably has 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety of the oligomer is preferably a lower alkyl polyalkyleneglycol moiety such as a polyethylene glycol moiety, a polypropyleneglycol moiety, or a polybutylene glycol moiety. When the polyalkyleneglycol moiety is a polypropylene glycol moiety, the moiety preferablyhas a uniform (i.e., not random) structure. An exemplary polypropyleneglycol moiety having a uniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

The oligomer may further comprise one or more additional hydrophilicmoieties (i.e., moieties in addition to the polyalkylene glycol moiety)including, but not limited to, sugars, polyalkylene glycols, andpolyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will beconsidered to be the same moiety if they are coupled by ether bonds. Forexample, the moiety—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—is a single polyethylene glycol moiety having six polyethylene glycolsubunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

is a polyethylene glycol moiety having four polyethylene glycol subunitsand an additional hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and no additionalhydrophilic moieties.

The oligomer preferably further comprises one or more lipophilicmoieties as will be understood by those skilled in the art. Thelipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbon atoms. Thelipophilic moiety preferably has between a lower limit of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upperlimit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. The lipophilicmoiety more preferably has between a lower limit of 2, 3, 4, 5, 6, 7, 8,9, or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilic moiety even morepreferably has between a lower limit of 3, 4, 5, 6, 7, 8, or 9 and anupper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thelipophilic moiety still more preferably has between a lower limit of 3,4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10 carbon atoms. Thelipophilic moiety most preferably has 6 carbon atoms. The lipophilicmoiety is preferably selected from the group consisting of saturated orunsaturated, linear or branched alkyl moieties, saturated orunsaturated, linear or branched fatty acid moieties, cholesterol, andadamantane. Exemplary alkyl moieties include, but are not limited to,saturated, linear alkyl moieties such as methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl;saturated, branched alkyl moieties such as isopropyl, sec-butyl,tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derivedfrom the above saturated alkyl moieties including, but not limited to,vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary fatty acid moieties include, but are not limited to,unsaturated fatty acid moieties such as lauroleate, myristoleate,palmitoleate, oleate, elaidate, erucate, linoleate, linolenate,arachidonate, eicosapentaentoate, and docosahexaenoate; and saturatedfatty acid moieties such as acetate, caproate, caprylate, caprate,laurate, arachidate, behenate, lignocerate, and cerotate.

The oligomer may filter comprise one or more spacer moieties as will beunderstood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theC-peptide polypeptide, to separate a first hydrophilic or lipophilicmoiety from a second hydrophilic or lipophilic moiety, or to separate ahydrophilic moiety or lipophilic moiety from a linker moiety. Spacermoieties are preferably selected from the group consisting of sugar,cholesterol and glycerine moieties. Sugar moieties may be various sugarmoieties as will be understood by those skilled in the art including,but not limited to, monosaccharide moieties and disaccharide moieties.Preferred monosaccharide moieties have between 4 and 6 carbon atoms.

The oligomer may further comprise one or more linker moieties that areused to couple the oligomer with the C-peptide polypeptide as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties. Thealkyl linker moiety may be a saturated or unsaturated, linear orbranched alkyl moiety as will be understood by those skilled in the artincluding, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Thealkoxy moiety may be various alkoxy moieties including, but not limitedto, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy,octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy,eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy,tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4, or 5and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moiety maybe a saturated or unsaturated, linear or branched fatty acid moiety aswill be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

The oligomer may further comprise one or more terminating moieties atthe one or more ends of the oligomer, which are not coupled to theC-peptide polypeptide. The terminating moiety is preferably an alkyl oralkoxy moiety. The alkyl or alkoxy moiety preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 and an upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbonatoms. The alkyl or alkoxy moiety more preferably has between a lowerlimit of 1, 2, 3, 4, 5, 6, or 7 and an upper limit of 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 carbon atoms. The alkyl or alkoxy moiety even morepreferably has between a lower limit of 1, 2, 3, 4, or 5 and an upperlimit of 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl or alkoxy moietystill more preferably has between a lower limit of 1, 2, 3, or 4 and anupper limit of 5, 6, or 7 carbon atoms. The alkyl moiety may be a linearor branched, saturated or unsaturated alkyl moiety as will be understoodby those skilled in the art including, but not limited to, methyl,ethyl, propyl, butyl, pentyl, hexyl heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,octadecyl, nonadecyl, eicosyl, isopropyl, sec-butyl, tert-butyl,2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,2-ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl,ethynyl, 1-propynyl, and 2-propynyl. The alkoxy moiety may be variousalkoxy moieties including, but not limited to, methoxy, ethoxy, propoxy,butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy,sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy,2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The terminating moiety ismore preferably a lower alkyl moiety such as methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl, or alower alkoxy moiety such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. Mostpreferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

According to other embodiments of the present invention, the oligomercomprises the structure of Formula XI:A-L_(j)-G_(k)-R-G′_(m)-R′-G″_(n)-T  (XI)wherein:

-   -   A is an activatable moiety;    -   L is a linker moiety,    -   G, G′ and G″ are individually selected spacer moieties;    -   R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,        or R′ is the lipophilic moiety and R is the polyalkylene glycol        moiety;    -   T is a terminating moiety; and    -   j, k, m and n are individually 0 or 1.

According to these embodiments of the present invention, thepolyalkylene glycol moiety has at least 1, 2, 3, 4, 5, 6 or 7polyalkylene glycol subunits. The polyalkylene glycol moiety preferablyhas between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more polyalkylene glycol subunits. The polyalkylene glycol moietymore preferably has between a lower limit of 2, 3, 4, 5, or 6 and anupper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 polyalkylene glycol subunits. Even more preferably, the polyalkyleneglycol moiety has between a lower limit of 3, 4, 5, or 6 and an upperlimit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol subunits. Thepolyalkylene glycol moiety still more preferably has between a lowerlimit of 4, 5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycolsubunits. The polyalkylene glycol moiety most preferably has 7polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene glycol moiety is apolypropylene glycol moiety, the moiety preferably has a uniform (i.e.,not random) structure. An exemplary polypropylene glycol moiety having auniform structure is as follows:

This uniform polypropylene glycol structure may be described as havingonly one methyl substituted carbon atom adjacent each oxygen atom in thepolypropylene glycol chain. Such uniform polypropylene glycol moietiesmay exhibit both lipophilic and hydrophilic characteristics.

According to these embodiments of the present invention, the lipophilicmoiety is a lipophilic moiety as will be understood by those skilled inthe art. The lipophilic moiety has at least 1, 2, 3, 4, 5, or 6 carbonatoms. The lipophilic moiety preferably has between a lower limit of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Thelipophilic moiety more preferably has between a lower limit of 2, 3, 4,5, 6, 7, 8, 9, or 10 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or carbon atoms. The lipophilicmoiety even more preferably has between a lower limit of 3, 4, 5, 6, 7,8, or 9 and an upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14carbon atoms. The lipophilic moiety still more preferably has between alower limit of 3, 4, 5, 6, or 7 and an upper limit of 6, 7, 8, 9, or 10carbon atoms. The lipophilic moiety most preferably has 6 carbon atoms.The lipophilic moiety is preferably selected from the group consistingof saturated or unsaturated, linear or branched alkyl moieties,saturated or unsaturated, linear or branched fatty acid moieties,cholesterol, and adamantane. Exemplary alkyl moieties include, but arenot limited to, saturated, linear alkyl moieties such as methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,nonadecyl and eicosyl; saturated, branched alkyl moieties such asisopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; andunsaturated alkyl moieties derived from the above saturated alkylmoieties including, but not limited to, vinyl, allyl, 1-butenyl,2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. Exemplary fatty acidmoieties include, but are not limited to, unsaturated fatty acidmoieties such as lauroleate, myristoleate, palmitoleate, oleate,elaidate, erucate, linoleate, linolenate, arachidonate,eicosapentaentoate, and docosahexaenoate; and saturated fatty acidmoieties such as acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate.

According to these embodiments of the present invention, the spacermoieties, G, G′ and G″, are spacer moieties as will be understood bythose skilled in the art. Spacer moieties are preferably selected fromthe group consisting of sugar moieties, cholesterol and glycerinemoieties. Sugar moieties may be various sugar moieties as will beunderstood by those skilled in the art including, but not limited to,monosaccharide moieties and disaccharide moieties. Preferredmonosaccharide moieties have between 4 and 6 carbon atoms. Preferably,oligomers of these embodiments do not include spacer moieties (i.e., k,m and n are preferably 0).

According to these embodiments of the present invention, the linkermoiety, L, may be used to couple the oligomer with the C-peptidepolypeptide as will be understood by those skilled in the art. Linkermoieties are preferably selected from the group consisting of alkyl andfatty acid moieties. The alkyl linker moiety may be a saturated orunsaturated, linear or branched alkyl moiety as will be understood bythose skilled in the art including, but not limited to, methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,nonadecyl eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl ethynyl, 1-propynyl,and 2-propynyl. The alkoxy moiety may be various alkoxy moietiesincluding, but not limited to, methoxy, ethoxy, propoxy, butoxy,pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy,sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy,2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy,2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy,ethynyloxy, 1-propynyloxy, and 2-propynyloxy. The alkyl linker moietymay have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 carbon atoms, and preferably has between 1, 2, 3, 4, or 5and 8, 9, 10, 11, or 12 carbon atoms. The fatty acid linker moiety maybe a saturated or unsaturated, linear or branched fatty acid moiety aswill be understood by those skilled in the art including, but notlimited to, lauroleate, myristoleate, palmitoleate, oleate, elaidate,erucate, linoleate, linolenate, arachidonate, eicosapentaentoate,docosahexaenoate, acetate, caproate, caprylate, caprate, laurate,arachidate, behenate, lignocerate, and cerotate. The fatty acid linkermoiety may have between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 carbon atoms and preferably has between 1, 2, 3,4, or 5 and 8, 10, 12, 14 or 16 carbon atoms.

According to these embodiments of the present invention, the terminatingmoiety, T, is preferably an alkyl or alkoxy moiety. The alkyl or alkoxymoiety preferably has between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 carbon atoms. The alkyl or alkoxy moietymore preferably has between a lower limit of 1, 2, 3, 4, 5, 6, or 7 andan upper limit of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Thealkyl or alkoxy moiety even more preferably has between a lower limit of1, 2, 3, 4, or 5 and an upper limit of 5, 6, 7, 8, 9, or 10 carbonatoms. The alkyl or alkoxy moiety still more preferably has between alower limit of 1, 2, 3, or 4 and an upper limit of 5, 6, or 7 carbonatoms. The alkyl moiety may be various linear or branched, saturated orunsaturated alkyl moieties as will be understood by those skilled in theart including, but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, eicosyl,isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl,allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl.Exemplary alkoxy moieties may be various alkoxy moieties including, butnot limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy,heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy,tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy,nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy,2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy,2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy,2-butenyloxy, ethynyloxy, 1-propynyloxy, and 2-propynyloxy. Theterminating moiety is more preferably a lower alkyl moiety such asmethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,or tert-pentyl, or a lower alkoxy moiety such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, ortert-pentyloxy. Most preferably, the terminating moiety is methyl ormethoxy. While the terminating moiety is preferably an alkyl or alkoxymoiety, it is to be understood that the terminating moiety may bevarious moieties as will be understood by those skilled in the artincluding, but not limited to, sugar moieties, cholesterol, alcohols,and fatty acid moieties.

According to these embodiments of the present invention, the activatablemoiety, A, is a moiety that allows for the coupling of the oligomer toan activating agent to form an activated oligomer capable of couplingwith the proinsulin polypeptide. The activatable moiety may be variousactivatable moieties as will be understood by those skilled in the artincluding, but not limited to, —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH,and NH₂.

In still other embodiments, the oligomer comprises the structure ofFormula XII:A-X(CH₂)_(m)Y(C₂H₄O)_(n)R  (XII)wherein:

-   -   A is —C(O)OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH₂;    -   X is an oxygen atom or a covalent bond, with the proviso X is        not an oxygen atom when A is —OH;    -   Y is an ester, an ether, a carbamate, a carbonate, or an amide        bonding moiety, and is preferably an ether bonding moiety,    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 1, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In still other embodiments, the oligomer comprises the structure ofFormula XIII:A—(CH₂)_(m)(OC₂H₄)_(n)OR  (XIII)wherein:

-   -   A is —C(O)—OH, C(S)—OH, —C(S)—SH, —OH, —SH, or NH₂;    -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In yet other embodiments, the oligomer comprises the structure ofFormula XIV:

wherein:

-   -   m is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 4,        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, or 30, is more preferably        between a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an        upper limit of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, 19, 20, 21, or 22, is even more preferably between a lower        limit of 3, 4, 5, 6, 7, 8, or 9 and an upper limit of 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, or 14, and is still more preferably has        between a lower limit of 3, 4, 5, 6, or 7 and an upper limit of        6, 7, 8, 9, or 10;    -   n is between a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, or 20 and an upper limit of 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,        43, 44, 45, 46, 47, 48, 49, or 50, is more preferably between a        lower limit of 2, 3, 4, 5, or 6 and an upper limit of 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is even        more preferably between a lower limit of 3, 4, 5, or 6 and an        upper limit of 5, 6, 7, 8, 9, 10, 11, or 12 polyalkylene glycol        subunits, is still more preferably between a lower limit of 4,        5, or 6 and an upper limit of 6, 7, or 8 polyalkylene glycol        subunits, and is most preferably 7; and    -   R is an alkyl moiety, a sugar moiety, cholesterol, adamantane,        an alcohol moiety, or a fatty acid moiety. The alkyl moiety may        be a linear or branched, saturated or unsaturated alkyl moieties        as will be understood by those skilled in the art including, but        not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,        heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,        tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl,        eicosyl, isopropyl, sec-butyl, tert-butyl, 2-methylbutyl,        tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl,        2-propylpentyl, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl,        1-propynyl, and 2-propynyl. The alkyl moiety is more preferably        a lower alkyl moiety such as methyl, ethyl, propyl, isopropyl,        butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. The alkyl        moiety is still more preferably a C₁ to C₃ alkyl. The alkyl        moiety is most preferably methyl. The fatty acid moiety may be a        saturated or unsaturated, linear or branched fatty acid moiety        as will be understood by those skilled in the art including, but        not limited to, lauroleate, myristoleate, palmitoleate, oleate,        elaidate, erucate, linoleate, linolenate, arachidonate,        eicosapentaentoate, docosahexaenoate, acetate, caproate,        caprylate, caprate, laurate, arachidate, behenate, lignocerate,        and cerotate.

In the various embodiments of method for synthesizing C-peptidepolypeptide oligomer conjugates described above, the oligomer iscovalently coupled to the C-peptide polypeptide. In some embodiments,the oligomer is coupled to the C-peptide polypeptide utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide a C-peptide polypeptide-oligomer conjugate thatacts as a prodrug. In certain instances, for example where the C-peptidepolypeptide-oligomer conjugate is biologically inactive (i.e., theconjugate lacks the ability to affect the body through the C-peptidepolypeptide's primary mechanism of action), a hydrolyzable coupling mayprovide for a time-release or controlled-release effect, providing thebiologically active C-peptide polypeptide over a given time period asone or more oligomers are cleaved from their respective biologicallyinactive C-peptide polypeptide-oligomer conjugates to provide thebiologically active C-peptide polypeptide. In other embodiments, theoligomer is coupled to the C-peptide polypeptide utilizing anon-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use ofa non-hydrolyzable bond may be preferable when it is desirable to allowthe biologically inactive C-peptide polypeptide-oligomer conjugate tocirculate in the bloodstream for an extended period of time, preferablyat least 2 hours. When the oligomer is coupled to the C-peptidepolypeptide utilizing a bonding moiety that comprises a carbonyl moiety,such as an ester, a carbamate, a carbonate, or an amide bonding moiety,the resulting C-peptide polypeptide-oligomer conjugate is a C-peptidepolypeptide-acyl oligomer conjugate.

Oligomers employed in the various methods of synthesizing C-peptidepolypeptide-oligomer conjugates described above are commerciallyavailable or may be synthesized by various methods as will be understoodby those skilled in the art. For example, polydispersed oligomers may besynthesized by the methods provided in one or more of the followingreferences: U.S. Pat. No. 4,179,337 to Davis et al.; U.S. Pat. No.5,567,422 to Greenwald; U.S. Pat. No. 5,359,030 to Ekwuribe; U.S. Pat.No. 5,438,040 to Ekwuribe, U.S. Pat. No. 5,681,811 to Ekwuribe, U.S.Pat. No. 6,309,633 to Ekwuribe et al. Non-polydispersed (e.g.,substantially monodispersed and monodispersed) oligomers may besynthesized by methods provided in one or more of the followingreferences: U.S. patent application Ser. No. 09/873,731 filed Jun. 4,2001 by Ekwuribe et al. entitled “Methods of Synthesizing SubstantiallyMonodispersed Mixtures of Polymers Having Polyethylene Glycol Mixtures”;U.S. patent application Ser. No. 09/873,797 filed Jun. 4, 2001 byEkwuribe et al. entitled “Mixtures of Drug-Oligomer ConjugatesComprising Polyalkylene Glycol, Uses Thereof, and Methods of MakingSame”; and U.S. patent application Ser. No. 09/873,899 filed Jun. 4,2001 by Ekwuribe et al. entitled “Mixtures of Insulin Drug-OligomerConjugates Comprising Polyalkylene Glycol, Uses Thereof, and Methods ofMaking Same”. Oligomers according to embodiments of the presentinvention are preferably substantially monodispersed and are morepreferably monodispersed. Exemplary methods for synthesizing preferredmonodispersed oligomers are provided in Examples 1 through 10 below.

The contacting of the pro-C-peptide polypeptide with the oligomer underconditions sufficient to provide a pro-C-peptide polypeptide-oligomerconjugate may be performed utilizing various conditions as will beunderstood by those skilled in the art. Preferably, the contacting ofthe pro-C-peptide polypeptide with the oligomer under conditionssufficient to provide a pro-C-peptide polypeptide-oligomer conjugatecomprises contacting the oligomer _with an activating agent underconditions sufficient to provide an activated oligomer; and contactingthe activated oligomer with the pro-C-peptide polypeptide underconditions sufficient to provide the pro-C-peptide polypeptideconjugate. The activated oligomer may be formed ex situ or in situ.

The activating agent may be various activating agents capable ofactivating one or more of the oligomers described above so that theoligomer is capable of reacting with nucleophilic hydroxyl functionsand/or amino functions in the proinsulin polypeptide as will beunderstood by those skilled in the art including, but not limited to,N-hydroxysuccinimide, p-nitrophenyl chloroformate,1,3-dicyclohexylcarbodiimide, and hydroxybenzotriazide.

One skilled in the art will understand the conditions sufficient tocouple the activating agent to the oligomer to provide an activatedoligomer. For example, one skilled in the art can refer to R. C. Larock,COMPRENSIVE ORGANIC TRANSFORMATIONS. A GUIDE TO FUNCTIONAL GROUPPREPARATIONS (2d Edition, N.Y., Wiley-VCH, 1999).

The conditions sufficient to couple the activated oligomer to thepro-C-peptide polypeptide will be understood to one of skill in the art.For example, the pro-C-peptide polypeptide may be dissolved in a dipolaraprotic solvent, such as dimethylsulfoxide, to provide a pro-C-peptidepolypeptide solution. A buffering agent, such as triethylamine, may beadded to the pro-C-peptide polypeptide solution. The activated oligomerdissolved in an anydrous solvent such as acetonitrile may then be addedto the pro-C-peptide polypeptide solution. One skilled in the art mayalso refer to R. C. Larock, COMREHENSIVE ORGANIC TRANSFORMATIONS. AGUIDE TO FUNCTIONAL GROUP PREPARATIONS (2d Edition, N.Y., Wiley-VCH,1999). The molar ratio of activated oligomer to pro-C-peptidepolypeptide is preferably greater than about 1:1, is more preferablygreater than about 2:1, is even more preferably greater than about 3:1,is still more preferably greater than about 4:1, and is still even morepreferably greater than about 5:1.

In the various embodiments of methods for synthesizing C-peptidepolypeptide-oligomer conjugates described above, more than one oligomer(i.e., a plurality of oligomers) may be coupled to the C-peptidepolypeptide portion of the pro-C-peptide polypeptide. The oligomers inthe plurality are preferably the same. However, it is to be understoodthat the oligomers in the plurality may be different from one another,or, alternatively, some of the oligomers in the plurality may be thesame and some may be different. When a plurality of oligomers arecoupled to the C-peptide polypeptide portion of the pro-C-peptidepolypeptide, it may be preferable to couple one or more of the oligomersto the C-peptide polypeptide portion of the pro-C-peptide polypeptidewith hydrolyzable bonds and couple one or more of the oligomers to theC-peptide polypeptide portion of the pro-C-peptide polypeptide withnon-hydrolyzable bonds. Alternatively, all of the bonds coupling theplurality of oligomers to the C-peptide polypeptide portion of thepro-C-peptide polypeptide may be hydrolyzable, but have varying degreesof hydrolyzability such that, for example, one or more of the oligomersis rapidly removed from the C-peptide polypeptide or C-peptidepolypeptide portion of the pro-C-peptide polypeptide by hydrolysis inthe body and one or more of the oligomers is slowly removed from theC-peptide polypeptide or C-peptide polypeptide portion by hydrolysis inthe body.

In the various embodiments of methods for synthesizing C-peptidepolypeptide-oligomer conjugates described above, the oligomer may becoupled to the C-peptide polypeptide portion of the pro-C-peptidepolypeptide at various nucleophilic residues of the C-peptidepolypeptide portion including, but not limited to, nucleophilic hydroxylfunctions and/or amino functions. A nucleophilic hydroxyl function maybe found, for example, at serine and/or tyrosine residues, and anucleophilic amino function may be found, for example, at histidineand/or lysine residues, and/or at the one or more N-termini of thepolypeptide. When an oligomer is coupled to the one or more N-termini ofthe proinsulin polypeptide, the coupling preferably forms a secondaryamine.

The cleaving of the one or more peptides from the pro-C-peptidepolypeptide-oligomer conjugate to provide the C-peptidepolypeptide-oligomer conjugate may be performed by various processes aswill be understood by those skilled in the art. Preferably, the cleavingof the one or more peptides from the pro-C-peptide polypeptide-oligomerconjugate comprises contacting the pro C-peptide polypeptide-oligomerconjugate with one or more enzymes that are capable of cleaving thebond(s) between the one or more peptides and the C-peptide polypeptideunder conditions sufficient to cleave the one or more peptides from thepro-C-peptide polypeptide-oligomer conjugate. As described in variousreferences, for example, Kemmler et al. “Studies on the Conversion ofProinsulin to Insulin,” J. Biol. Chem., 246:6786-6791 (1971), oneskilled in the art will understand how to select appropriate enzymes inview of the particular peptide bond(s) to be cleaved and how to provideconditions sufficient to cleave the one or more peptides from thepro-C-peptide polypeptide-oligomer conjugate. The one or more enzymespreferably comprise various enzymes including, but not limited to,trypsin, chymotrypsin, carboxy peptidase B, and mixtures thereof. Morepreferably, the one or more enzymes are selected from the groupconsisting of trypsin, carboxy peptidase B, and mixtures thereof 115

The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Example 1 Synthesis of6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)ethoxy]-ethoxy}-ethoxy)hexanoicacid 2,5-dioxo-pyrrolidin-1-yl ester (8)

Hexaethylene glycol monobenzyl ether (1). An aqueous sodium hydroxidesolution prepared by dissolving 3.99 g (100 mmol) NaOH in 4 ml water wasadded slowly to monodispersed hexaethylene glycol (28.175 g, 25 ml, 100mmol). Benzyl chloride (3.9 g, 30.8 mmol, 3.54 ml) was added and thereaction mixture was heated with stirring to 100° C. for 18 hours. Thereaction mixture was then cooled, diluted with brine (250 ml) andextracted with methylene chloride (200 ml×2). The combined organiclayers were washed with brine once, dried over Na₂SO₄, filtered andconcentrated in vacuo to a dark brown oil.

The crude product mixture was purified via flash chromatography (silicagel, gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) toyield 8.099 g (70%) of monodispersed compound 1 as a yellow oil.

Ethyl 6-methylsulfonyloxyhexanoate (2). A solution of monodispersedethyl 6-hydroxyhexanoate (50.76 m; 50.41 g, 227 mmol) in drydichloromethane (75 ml) was chilled in an ice bath and placed under anitrogen atmosphere. Triethylamine (34.43 ml, 24.99 g, 247 mmol) wasadded. A solution of methanesulfonyl chloride (19.15 ml, 28.3 g, 247mmol) in dry dichloromethane (75 ml) was added dropwise from an additionfunnel. The mixture was stirred for three and one half hours, slowlybeing allowed to come to room temperature as the ice bath melted. Themixture was filtered through silica gel and the filtrate was washedsuccessively with water, saturated NaHCO₃, water and brine. The organicswere dried over Na₂SO₄, filtered and concentrated in vacuo to a paleyellow oil. Final purification of the crude product was achieved byflash chromatography (silica gel, 1/1 hexanes/ethyl acetate) to give themonodispersed compound 2 (46.13 g, 85%) as a clear, colorless oil. FABMS: m/e 239 (M+H), 193 (M−C₂H₅O).

6-{2-[2-(2-{2-[2-(2-Benzyloxyethoxy)ethoxy]ethoxy}-ethoxy)ethoxy]-ethozy}-hexanoic acid ethyl ester (3). Sodium hydride (3.225 gor a 60% oil dispersion, 80.6 mmol) was suspended in 80 ml of anhydroustoluene, placed under a nitrogen atmosphere and cooled in an ice bath. Asolution of the monodispersed alcohol 9 (27.3 g, 73.3 mmol) in 80 ml drytoluene was added to the NaH suspension. The mixture was stirred at 0°C. for thirty minutes, allowed to come to room temperature and stirredfor another five hours, during which time the mixture became a clearbrown solution. The monodispersed mesylate 10 (19.21 g, 80.6 mmol) in 80ml dry toluene was added to the NaH/alcohol mixture, and the combinedsolutions were stirred at room temperature for three days. The reactionmixture was quenched with 50 ml methanol and filtered through basicalumina. The filtrate was concentrated in vacuo and purified by flashchromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanesto ethyl acetate) to yield the monodispersed compound 3 as a pale yellowoil (16.52 g, 44%). FAB MS: m/e 515 (M+H).

6-{2-[2-(2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]ethoxy}-hexanoicacid ethyl ester (4). Substantially monodispersed benzyl ether 3 (1.03g, 2.0 mmol) was dissolved in 25 ml ethanol. To this solution was added270 mg 10% Pd/C, and the mixture was placed under a hydrogen atmosphereand stirred for four hours, at which time TLC showed the completedisappearance of the starting material. The reaction mixture wasfiltered through Celite 545 to remove the catalyst, and the filtrate wasconcentrated in vacuo to yield the monodispersed compound 4 as a clearoil (0.67 g, 79%). FAB MS: m/e 425 (M+H), 447 (M+Na).

6-{2-[2-(2-{2-[2-(2-methylsulfonylethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoicacid ethyl ester (5). The monodispersed alcohol 4 (0.835 g, 1.97 mmol)was dissolved in 3.5 ml dry dichlorometane and placed under a nitrogenatmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added andthe mixture was chilled in an ice bath. After two minutes, themethanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. Themixture was stirred for 15 minutes at 0° C., then at room temperaturefor two hours. The reaction mixture was filtered through silica gel toremove the triethylammonium chloride, and the filtrate was washedsuccessively with water, saturated NaHCO₃, water and brine. The organicswere dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, 9/1 ethylacetate/methanol) to give monodispersed compound 5 as a clear oil (0.819g, 83%). FAB MS: m/e 503 (M+H).

6-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethozy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)hexanoicacid ethyl ester (6). NaH (88 mg of a 60% dispersion in oil, 2.2 mmol)was suspended in anhydrous toluene (3 ml) under N₂ and chilled to 0° C.Monodispersed diethylene glycol monomethyl ether (0.26 ml, 0.26 g, 2.2mmol) that had been dried via azeotropic distillation with toluene wasadded. The reaction mixture was allowed to warm to room temperature andstirred for four hours, during which time the cloudy grey suspensionbecame clear and yellow and then turned brown. Mesylate 5 (0.50 g, 1.0mmol) in 2.5 ml dry toluene was added. After stirring at roomtemperature over night, the reaction was quenched by the addition of 2ml of methanol and the resultant solution was filtered through silicagel. The filtrate was concentrated in vacuo and the FAB MS: m/e 499(M+H), 521 (M+Na). Additional purification by preparatory chromatography(silica gel, 19/3 chloroform/methanol) provided the monodispersedcompound 6 as a clear yellow oil (0.302 g 57%). FAB MS: m/e 527 (M+H),549 (M+Na).

6-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)ethozy]-ethoxy}-ethoxy)hexanoicacid (7). Monodispersed ester 6 (0.25 g, 0.46 mmol) was stirred for 18hours in 0.71 ml of 1 N NaOH. After 18 hours, the mixture wasconcentrated in vacuo to remove the alcohol and the residue dissolved ina further 10 ml of water. The aqueous solution was acidified to pH 2with 2 N HCl and the product was extracted into dichloromethane (30ml×2). The combined organics were then washed with brine (25 ml×2),dried over Na₂SO₄, filtered and concentrated in vacuo to yield themonodispersed compound 15 as a yellow oil (0.147 g, 62%). FAB MS: m/e499 (M+H), 521 (M+Na).

6-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)ethoxy]-ethoxy}-ethoxy)hexanoicacid 2,5-dioxo-pyrrolidin-1-yl ester (8). Monodispersed acid 7 (0.209 g,0.42 mmol) was dissolved in 4 ml of dry dichloromethane and added to adry flask already containing NHS (N-hydroxysuccinimide) (57.8 mg, 0.502mmol) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride) (98.0 mg, 0.502 mmol) under a N₂ atmosphere. The solutionwas stirred at room temperature overnight and filtered through silicagel to remove excess reagents and the urea formed from the EDC. Thefiltrate was concentrated in vacuo to provide the activatedmonodispersed oligomer 8 as a dark yellow oil (0.235 g, 94%). FAB MS:m/e 596 (M+H), 618 (M+Na).

Example 2 Synthesis of Activated MPEG₇-C₈ (14)

Mesylate of triethylene glycol monomethyl ether (9). To a solution ofCH₂Cl₂ (100 mL) cooled to 0° C. in an ice bath was added monodispersedtriethylene glycol monomethyl ether (25 g, 0.15 mol). Then triethylamine(29.5 ml, 0.22 mol) was added and the solution was stirred for 15 min at0° C., which was followed by dropwise addition of methanesulfonylchloride (13.8 mL, 0.18 mol, dissolved in 20 mL CH₂Cl₂). The reactionmixture was stirred for 30 min at 0° C., allowed to warm to roomtemperature, and then stirred for 2 h. The crude reaction mixture wasfiltered through Celite (washed CH₂Cl_(2˜)200 mL), then washed with H₂O(300 mL), 5% NaHCO₃ (300 mL), H₂O (300 mL), sat NaCl (300 mL), driedMgSO₄, and evaporated to dryness. The oil was then placed on a vacuumline for 2 h to ensure dryness and afforded the monodispersed compound 9as a yellow oil (29.15 g, 80% yield).

Heptaethylene glycol monomethyl ether (10). To a solution ofmonodispersed tetraethylene glycol (51.5 g, 0.27 mol) in THE (1L) wasadded potassium t-butoxide (14.8 g, 0.13 mol, small portions over ˜30min). The reaction mixture was then stirred for 1 h and then 9 (29.15 g,0.12 mol) dissolved in THF (90 mL) was added dropwise and the reactionmixture was stirred overnight. The crude reaction mixture was filteredthrough Celite (washed CH₂Cl₂, ˜200 mL) and evaporated to dryness. Theoil was then dissolved in HCl (250 mL, 1 N) and washed with ethylacetate (250 mL) to remove excess 9. Additional washings of ethylacetate (125 mL) may be required to remove remaining 9. The aqueousphase was washed repetitively with CH₂Cl₂ (125 mL volumes) until most ofthe compound 18 has been removed from the aqueous phase. The firstextraction will contain 9, 10, and dicoupled side product and should beback extracted with HCl (125 mL, 1N). The organic layers were combinedand evaporated to dryness. The resultant oil was then dissolved inCH₂Cl₂ (100 mL) and washed repetitively with H₂O (50 mL volumes) until10 was removed. The aqueous fractions were combined, total volume 500mL, and NaCl was added until the solution became cloudy and then waswashed with CH₂Cl₂ (2×500 mL). The organic layers were combined, driedMgSO₄, and evaporated to dryness to afford the monodispersed compound 10as an oil (16.9 g, 41% yield). It may be desirable to repeat one or moresteps of the purification procedure to ensure high purity.

8-Bromooctoanate (11). To a solution of monodispersed 8-bromooctanoicacid (5.0 g, 22 mmol) in ethanol (100 mL) was added H₂SO₄ (0.36 mL, 7.5mmol) and the reaction was heated to reflux with stirring for 3 h. Thecrude reaction mixture was cooled to room temperature and washed H₂O(100 mL), sat. NaHCO₃ (2×100 mL), H₂O (100 mL), dried MgSO₄, andevaporated to dryness to afford a clear oil 11 (5.5 g, 98% yield).

MPEG₇-C₈ ester (12). To a solution of the monodispersed compound 10 (3.0g, 8.8 mmol) in ether (90 mL) was added potassium t-butoxide (1.2 g, 9.6mmol) and the reaction mixture was stirred for 1 h. Then dropwiseaddition of the monodispersed compound 11 (2.4 g, 9.6 mmol), dissolvedin ether (10 mL), was added and the reaction mixture was stirredovernight. The crude reaction mixture was filtered through Celite(washed CH₂Cl₂, ˜200 mL) and evaporated to dryness. The resultant oilwas dissolved in ethyl acetate and washed H₂O (2×200 mL), dried MgSO₄,and evaporated to dryness. Column chromatography (Silica, ethyl acetateto ethyl acetate/methanol, 10:1) was performed and afforded themonodispersed compound 12 as a clear oil (0.843 g, 19% yield).

MPEG₇C₈ acid (13). To the oil of the monodispersed compound 12 (0.70 g,1.4 mmol) was added 1N NaOH (2.0 mL) and the reaction mixture wasstirred for 4 h. The crude reaction mixture was concentrated, acidified(pH˜2), saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). The organiclayers were combined, washed sat. NaCl, dried MgSO₄, and evaporated todryness to afford the monodispersed compound 13 as a clear oil (0.35 g,53% yield).

Activation of MPEG₇C₈ acid. Monodispersed mPEG7-C8-acid 13 (0.31 g, 0.64mmol) was dissolved in 3 ml of anhydrous methylene chloride and thensolution of N-hydroxysuccinimide (0.079 g, 0.69 mmol) and EDCI HCl(135.6 mg, 0.71 mmol) in anhydrous methylene chloride added. Reactionwas stirred for several hours, then washed with 1N HCl, water, driedover MgSO₄, filtered and concentrated. Crude material was purified bycolumn chromatography, concentrated to afford monodispersed activatedMPEG₇-C₈ 14 as a clear oil and dried via vacuum.

Example 3 Synthesis of Activated MPEG₇-C₁₀ (19)

10-hydroxydecanoate (15). To a solution of monodispersed10-hydroxydecanoic acid (5.0 g, 26.5 mmol) in ethanol (100 mL) was addedH₂SO₄ (0.43 mL, 8.8 mmol) and the reaction was heated to reflux withstirring for 3 h. The crude reaction mixture was cooled to roomtemperature and washed H₂O(100 mL), sat. NaHCO₃ (2×100 mL), H₂O (100mL), dried MgSO₄, and evaporated to dryness to afford the monodispersedcompound 15 as a clear oil (6.9 g, 98% yield).

Mesylate of 10-hydroxydecanoate (16). To a solution of CH₂Cl₂ (27 mL)was added monodispersed 10-hydroxydecanoate 15 (5.6 g, 26 mmol) andcooled to 0° C. in an ice bath. Then triethylamine (5 mL, 37 mmol) wasadded and the reaction mixture was stirred for 15 min at 0° C. Thenmethanesulfonyl chloride (2.7 mL, 24 mmol) dissolved in CH₂Cl₂ (3 mL)was added and the reaction mixture was stirred at 0° C. for 30 min, theice bath was removed and the reaction was stirred for an additional 2 hat room temperature. The crude reaction mixture was filtered throughCelite (washed CH₂Cl₂, 80 mL) and the filtrate was washed H₂O (100 mL),5% NaHCO₃ (2×100 mL), H₂O (100 mL), sat NaCl (100 mL), dried MgSO₄, andevaporated to dryness to afford the monodispersed compound 16 as ayellowish oil (7.42 g, 97% yield).

MPEG₇C₁₀ Ester (17). To a solution of substantially monodispersedheptaethylene glycol monomethyl ether 10 (2.5 g, 7.3 mmol) intetrhydrofuran (100 mL) was added sodium hydride (0.194 g, 8.1 mmol) andthe reaction mixture was stirred for 1 h. Then dropwise addition ofmesylate of monodispersed 10-hydroxydecanoate 16 (2.4 g, 8.1 mmol),dissolved in tetrahydrofuran (10 mL), was added and the reaction mixturewas stirred overnight. The crude reaction mixture was filtered throughCelite (washed CH₂Cl₂, 200 mL) and evaporated to dryness. The resultantoil was dissolved in ethyl acetate and washed H₂O (2×200 mL), driedMgSO₄, evaporated to dryness, chromatographed (silica, ethylacetate/methanol, 10:1), and chromatographed (silica, ethyl acetate) toafford the monodispersed compound 17 as a clear oil (0.570 g, 15%yield).

MPEG₇-C₁₀ Acid (18). To the oil of monodispersed mPEG₇-C₁₀ ester 17(0.570 g, 1.1 mmol) was added 1N NaOH (1.6 mL) and the reaction mixturewas stirred overnight. The crude reaction mixture was concentrated,acidified (pH˜2), saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). Theorganic layers were combined, washed sat. NaCl (2×50 mL), dried MgSO₄,and evaporated to dryness to afford the monodispersed compound 18 as aclear oil (0.340 g, 62% yield).

Activation of MPEG₇-C₁₀ Acid. The monodispersed acid 18 was activatedusing procedures similar to those described above in Example 10 toprovide activated MPEG₇-C₁₀ Oligomer 19.

Example 4 Synthesis of Activated C₁₈(PEG₆) Oliomer (22)

Synthesis of Cl₈(PEG₆) Oligomer (20). Monodispersed stearoyl chloride(0.7 g, 2.31 mmol) was added slowly to a mixture of monodispersed PEG₆(5 g, 17.7 mmol) and pyridine (0.97 g, 12.4 mmol) in benzene. Thereaction mixture was stirred for several hours (5). The reaction wasfollowed by TLC using ethylacetate/methanol as a developing solvent.Then the reaction mixture was washed with water, dried over MgSO₄,concentrated and dried via vacuum. Purified monodispersed compound 20was analyzed by FABMS: m/e 549/M⁺H.

Activation of C₁₈(PEG₆) Oligomer. Activation of monodispersed C₁₈(PEG₆)oligomer was accomplished in two steps:

-   -   1) Monodispersed stearoyl-PEG₆ 20 (0.8 g, 1.46 mmol) was        dissolved in toluene and added to a phosgene solution (10 ml 20%        in toluene) which was cooled with an ice bath. The reaction        mixture was stirred for 1 h at 0° C. and then for 3 h at room        temperature. Then phosgene and toluene were distilled off and        the remaining substantially monodispersed stearoyl PEG6        chloroformate 21 was dried over P₂O₅ overnight.    -   2) To a solution of monodispersed stearoyl-PEG₆ chloroformate 21        (0.78 g, 1.27 mmol) and TEA (128 mg, 1.27 mmol) in anhydrous        methylene chloride, N-hydroxy succinimide (NHS) solution in        methylene chloride was added. The reaction mixture was stirred        for 16 hours, then washed with water, dried over MgSO₄,        filtered, concentrated and dried via vacuum to provide the        monodispersed activated C₁₈(PEG₆) oligomer 22.

Example 5 Synthesis of Activated C₁₈(PEG₈) Oligomer (28)

Tetraethylene glycol monobenzylether (23). To the oil of monodispersedtetraethylene glycol (19.4 g, 0.10 mol) was added a solution of NaOH(4.0 g in 4.0 mL) and the reaction was sired for 15 mm. Then benzylchloride (3.54 mL, 30.8 mmol) was added and the reaction mixture washeated to 100° C. and stirred overnight. The reaction mixture was cooledto room temperature, diluted with sat NaCl (250 mL), and washed CH₂Cl₂(2×200 mL). The organic layers were combined, washed sat. NaCl, driedMgSO₄, and chromatographed (silica, ethyl acetate) to afford themonodispersed compound 23 as a yellow oil (6.21 g, 71% yield).

Mesylate of tetraethylene glycol monobenzylether (24). To a solution ofCH₂Cl₂ (20 mL) was added monodispersed tetraethylene glycolmonobenzylether 23 (6.21 g, 22 mmol) and cooled to 0° C. in an ice bath.Then triethylamine (3.2 mL, 24 mmol) was added and the reaction mixturewas stirred for 15 min at 0° C. Then methanesulfonyl chloride (1.7 mL,24 mmol) dissolved in CH₂Cl₂ (2 mL) was added and the reaction mixturewas stirred at 0° C. for 30 min, the ice bath was removed and thereaction was stirred for an additional 2 h at room temperature. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, 80mL) and the filtrate was washed H₂O (100 mL), 5% NaHCO₃ (2×100 mL), H₂O(100 mL), sat. NaCl (100 mL), and dried MgSO₄. The resulting yellow oilwas chromatographed on a pad of silica containing activated carbon (10g) to afford the monodispersed compound 24 as a clear oil (7.10 g, 89%yield).

Octaethylene glycol monobenzylether (25). To a solution oftetrahydrofuran (140 mL) containing sodium hydride (0.43 g, 18 mmol) wasadded dropwise a solution of monodispersed tetraethylene glycol (3.5 g,18 mmol) in tetrahydrofuran (10 mL) and the reaction mixture was stirredfor 1 h. Then mesylate of monodispersed tetraethylene glycolmonobenzylether 24 (6.0 g, 16.5 mmol) dissolved in tetrahydrofuran (10mL) was added dropwise and the reaction mixture was stirred overnight.The crude reaction mixture was filtered through Celite (washed, CH₂Cl₂,250 mL) and the filtrate was washed H₂O, dried MgSO₄, and evaporated todryness. The resultant oil was chromatographed (silica, ethylacetate/methanol, 10:1) and chromatographed (silica,chloroform/methanol, 25:1) to afford the monodispersed compound 25 as aclear oil (2.62 g, 34% yield).

Synthesis of Stearate PEG₈-Benzyl (26). To a stirred cooled solution ofmonodispersed octaethylene glycol monobenzylether 25 (0.998 g, 2.07mmol) and pyridine (163.9 mg, 2.07 mmol) was added monodispersedstearoyl chloride (627.7 mg, 2.07 mmol) in benzene. The reaction mixturewas stirred overnight (18 hours). The next day the reaction mixture waswashed with water, dried over MgSO₄, concentrated and dried via vacuum.Then the crude product was chromatographed on flash silica gel column,using 10% methanol/90% chloroform. The fractions containing the productwere combined, F concentrated and dried via vacuum to afford themonodispersed compound 26.

Hydrogenolysis of Stearate-PEG₈-Benzyl. To a methanol solution ofmonodispersed stearate-PEG₈-Bzl26 (0.854 g 1.138 mmol) Pd/C(10%)(palladium, 10% wt. on activated carbon) was added. The reaction mixturewas stirred overnight (18 hours) under hydrogen. Then the solution wasfiltered, concentrated and purified by flash column chromatography using10% methanol/90% chloroform, fractions with R₁=0.6 collected,concentrated and dried to provide the monodispersed acid 27.

Activation of C₁₈(PEG₈) Oligomer. Two step activation of monodispersedstearate-PEG₈ oligomer 27 was performed as described for stearate-PEG₆in Example 4 above to provide the monodispersed activated C₁₈(PEG₈)oligomer 28.

Example 6 Synthesis of Activated Triethylene Glycol Monomethyl Oligomers

A solution of toluene containing 20% phosgene (100 ml, approximately18.7 g, 189 mmol phosgene) was chilled to 0° C. under a N₂ atmosphere.Monodispersed mTEG (triethylene glycol, monomethyl ether, 7.8 g, 47.5mmol) was dissolved in 25 mL anhydrous ethyl acetate and added to thechilled phosgene solution. The mixture was stirred for one hour at 0°C., then allowed to warm to room temperature and stirred for another twoand one half hours. The remaining phosgene, ethyl acetate and toluenewere removed via vacuum distillation to leave the monodispersed mTEGchloroformate as a clear oily residue.

The monodispersed nTEG chloroformate was dissolved in 50 mL of drydichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5mmol) and NHS (N-hydroxysuccinimide, 5.8 g, 50.4 mmol). The mixture wasstirred at room temperature under a dry atmosphere for twenty hoursduring which time a large amount of white precipitate appeared. Themixture was filtered to remove this precipitate and concentrated invacuo. The resultant oil was taken up in dichloromethane and washedtwice with cold deionized water, twice with 1N HCl and once with brine.The organics were dried over MgSO₄, filtered and concentrated to providethe monodispersed title compound as a clear, light yellow oil. Ifnecessary, the NHS ester could be further purified by flashchromatography on silica gel using EtOAc as the elutant.

Example 7 Synthesis of Activated Palmitate-TEG Oligomers

Monodispersed palmitic anhydride (5 g; 10 mmol) was dissolved in dry THF(20 mL) and stirred at room temperature. To the stirring solution, 3 molexcess of pyridine was added followed by monodispersed triethyleneglycol (1.4 mL). The reaction mixture was stirred for 1 hour (progressof the reaction was monitored by TLC; ethyl acetate-chloroform; 3:7). Atthe end of the reaction, THF was removed and the product was mixed with10% H₂SO₄ acid and extracted ethyl acetate (3×30 mL). The combinedextract was washed sequentially with water, brine, dried over MgSO₄, andevaporated to give monodispersed palmitate-TEG oligomers.

A solution of N,N′-disuccinimidyl carbonate (3 mmol) in DMF (˜10 mL) isadded to a solution of the monodispersed palmitate-TEG oligomers (1mmol) in 10 mL of anydrous DMF while sting. Sodium hydride (3 mmol) isadded slowly to the reaction mixture. The reaction mixture is stirredfor several hours (e.g., 5 hours). Diethyl ether is added to precipitatethe monodispersed activated title oligomer. This process is repeated 3times and the product is finally dried.

Example 8 Synthesis of Activated Hexaethylene Glycol MonomethylOligomers

Monodispersed activated hexaethylene glycol monomethyl ether wasprepared analogously to that of monodispersed triethylene glycol inExample 14 above. A 20% phosgene in toluene solution (35 mL, 6.66 g,67.4 mmol phosgene) was chilled under a N₂ atmosphere in an ice/saltwater bath. Monodispersed hexaethylene glycol (1.85 mL, 2.0 g, 6.74mmol) was dissolved in S mL anhydrous EtOAc and added to the phosgenesolution via syringe. The reaction mixture was kept stirring in the icebath for one hour, removed and stirred a further 2.5 hours at roomtemperature. The phosgene, EtOAc, and toluene were removed by vacuumdistillation, leaving monodispersed methyl hexaethylene glycolchloroformate as a clear, oily residue.

The monodispersed chloroformate was dissolved in 20 mL drydichloromethane and placed under a dry, inert atmosphere. Triethylamine(0.94 mL, 0.68 g, 6.7 mmol) and then NHS(N-hydroxy succinimide, 0.82 g,7.1 mmol) were added, and the reaction mixture was stirred at roomtemperature for 18 hours. The mixture was filtered through silica gel toremove the white precipitate and concentrated in vacuo. The residue wastaken up in dichloromethane and washed twice with cold water, twice with1 N HCl and once with brine. The organics-were dried over Na₂SO₄,filtered and concentrated. Final purification was done via flashchromatography (silica gel, EtOAc) to obtain the activated monodispersedhexaethylene monomethyl ether.

Example 9 Synthesis of Avtivated Heptaethylene Glycol Monomethyl Ether

8-Methoxy-1-(methylsulfonyl)oxy-3,6-dioxaoctane (29). A solution ofmonodispersed triethylene glycol monomethyl ether molecules (4.00 mL,4.19 g, 25.5 mmol) and triethylamine (4.26 mL, 3.09 g, 30.6 mmol) in drydichloromethane (50 mL) was chilled in an ice bath and place under anitrogen atmosphere. A solution of methanesulfonyl chloride (2.37 mL,3.51 g, 30.6 mmol) in dry dichloromethane (20 mL) was added dropwisefrom an addition funnel. Ten minutes after the completion of thechloride addition, the reaction mixture was removed from the ice bathand allowed to come to room temperature. The mixture was stirred for anadditional hour, at which time TLC (CHCl₃ with 15% MeOH as the elutant)showed no remaining triethylene glycol monomethyl ether.

The reaction mixture was diluted with another 75 mL of dichloromethaneand washed successively with saturated NaHCO₃, water and brine. Theorganics were dried over Na₂SO₄, filtered and concentrated in vacuo togive a monodispersed mixture of compounds 29 as a clear oil (5.31 g,86%).

Heptaethylene glycol mono methyl ether (30). To a stirred solution ofmonodispersed tetraethylene glycol (35.7 mmol) in dry DMF (25.7 mL),under N₂ was added in portion a 60% dispersion of NaH in mineral oil,and the mixture was stirred at room temperature for 1 hour. To theresulting sodium salt of the tetraethylene glycol was added a solutionof monodispersed mesylate 29 (23.36) in dry DMF (4 ml) in a singleportion, and the mixture was stirred at room temperature for 3.5 hours.Progress of the reaction was monitored by TLC (12% CH₃OH—CHCl₃). Thereaction mixture was diluted with an equal amount of 1N HC1, andextracted with ethyl acetate (2×20 ml) and discarded. Extraction ofaqueous solution and work-up gave monodispersed heptaethylene glycolmonomethyl ether 30 (82-84% yield). Oil; Rf 0.46(methanol:chloroform=3:22); MS m/z calc'd for C₁₅H₃₂O₈ 340.21 (M⁺+1),found 341.2.

Activation of heptaethylene glycol monomethyl ether. Monodispersedheptaethylene glycol monomethyl ether 30 is activated by a proceduresimilar to that used in Example 6 above to activate triethylene glycolmonomethyl ether to provide the activated heptaethylene glycolmonomethyl ether.

Example 10 Synthesis of Activated Decaethylene Glycol Monomethyl Ether(33)

20-methoxy-1-(methylsulfonyl)oxy-3,6,9,12,15,18-hexaoxaeicosane (31).Monodispersed compound 31 was obtained in quantitative yield fromcompound 30 and methanesulfonyl chloride as described for 29 in Example9 above, as an oil; Rf 0.4 (ethyl acetate:acetonitrile=1:5); MS m/zcalc'd for C₁₇H₃₇O₁₀ 433.21 (⁺+1), found 433.469.

Decaethylene glycol monomethyl ether (32). Monodispersed compound 32 wasprepared from compound 31 and monodispersed triethylene glycol using theprocedure described above in Example 17. Oil; Rf 0.41(methanol:chloroform=6:10); MS m/z calc'd for C₂₁H₄₄O₁₁ 472.29 (M⁺+1),found 472.29.

Activation of decaethylene glycol monomethyl ether. Monodisperseddecaethylene glycol monomethyl ether 32 is activated by a proceduresimilar to that used in Example 6 above to activate triethylene glycolmonomethyl ether to provide the activated decaethylene glycol monomethylether 33.

Example 11 Preparation of Lys^(B29)-Oligomer-Conjugated Insulin

Conjugation of Recombinant Proinsulin I. Recombinant Proinsulin I (MW10,642 Daltons) was obtained from Biobras, of Belo Horizonte, Brazil. A2.32×10⁻³ mmol portion of proinsulin I was dissolved in 10 mL of DMSO.To the solution was added 324 μL of triethylamine. The resultingsolution was allowed to stir for 5 minutes, and then a solution ofactivated methylheptaethylene glycol ((PEG7)-hexyl oligomer) (9.30×10⁻³mol) in acetonitrile was added. The course of the conjugation(acylation) reaction was monitored by HPLC. When reaction appeared to becomplete, it was quenched by addition of 3.54 mL of 5% aqueoustrifluoroacetic acid solution. The reaction mixture was then processedand exchanged into 100 mM Tris-HCl Buffer, pH 7.6. The HPLC profile ofthe product mixture, oligomer-conjugated recombinant Proinsulin I, isshown in FIG. 11.

(b) Enzyme Cocktail Cleavage of Oligomer-Conjugated RecombinantProinsulin I. An aliquot of the Tris-HCl solution of the product mixturefrom Example 1(a) was analyzed by HPLC to determine the polypeptideconcentration. A solution of trypsin (TPCK treated; from bovinepancreas) was prepared in 100 mM Tris-HCl Buffer, pH 7.6. A solution ofcarboxypeptidase B (from porcine pancreas) was prepared in 100 mMTris-HCl Buffer, pH 7.6. The product mixture from Example 11(a) (0.424μmol/mL) was then allowed to react with trypsin (5.97×10⁻³ μmol/mL) andcarboxypeptidase B (1.93×10⁻⁴ μmol/mL). After 30 minutes, the reactionwas quenched by the addition of 1.58 mL of 1% trifluoroacetic acid inacetonitrile. The major products were identified by HPLC retention time(relative to the retention times of known reference standards) and massspectral analysis. Insulin (10%) andLys^(B29)-Hexyl-PEG7-Oligomer-Conjugated Insulin (84%) were thusobtained (FIGS. 11-13).

Example 12 Isolation of the Products of Oligomer-Conjugation ofRecombinant Proinsulin I

Reversed-phase HPLC was used to isolate the major products from theproduct mixture obtained from the conjugation reaction described inExample 11(a). An HPLC column (1.0 cm. i.d.×25 cm. length) was packedwith a commercially available C18 stationary phase known to be usefulfor the separation of peptides and proteins, and then was incorporatedinto an HPLC system. The system was equilibrated with elution buffer, amixture comprising 72% mobile phase A (H₂O with 0.1% trifluoroaceticacid) and 28% mobile phase B (acetonitrile with 0.1% trifluoroaceticacid) that was delivered at a flow rate of 5 mL/min. A solution of theproduct mixture in 100 mM Tris-HCl Buffer, pH 7.6, was applied to thereversed-phase column, and the products were separated and eluted usinga gradient in which the acetonitrile component of the elution buffer(mobile phase B) was increased as follows:

-   -   28%-30% mobile phase B over 60 minutes, then    -   30%-32% mobile phase B over 30 minutes, then    -   32%-36% mobile phase B over 40 minutes.        Fractions were collected and individually analyzed by HPLC to        determine the identity and purity of the product contained        therein. Common fractions containing one of the four products        (monoconjugate-A (“Proinsulin I Monoconjugate-A”),        monoconjugate-B (“Proinsulin I Monoconjugate-B”), diconjugate        (“Proinsulin I Diconjugate”) and triconjugate (“Proinsulin I        Triconjugate”) were then pooled, and the solvent was removed by        rotary evaporation. HPLC (FIG. 14) and mass spectral analysis        were used to determine the identity and purity of each isolate.

Example 13 Enzyme Cocktail Cleavage of Isolated Conjugates ofRecombinant Proinsulin (I)

Each conjugate (Proinsulin I Mono A, Mono B, Di, or Tri) that wasisolated using the procedure described in Example 12 was dissolved in100 mM Tris-HCl Buffer, pH 7.6, and analytical HPLC was used todetermine the polypeptide concentration of the resulting solution. Asolution of trypsin (TPCK treated; from bovine pancreas) was prepared in100 mM Tris-HCl Buffer, pH 7.6. A solution of carboxypeptidase B (fromporcine pancreas) was prepared in 100 mM Tris-HCl Buffer, pH 7.6. Thecrude mixture (1 mmol) was then allowed to react with trypsin (1.39×10-3mmol) and carboxypeptidase B (4.56×10⁻⁴ mmol). After 30 minutes, thereaction was quenched by addition of 1% trifluoroacetic acid inacetonitrile. The product mixture from each reaction was processed andanalyzed by HPLC. The HPLC retention time relative to that of referencestandards and mass spectral analysis were used to determine the identityand purity of each product (Table 1).

TABLE 1 Oligomer-conjugates of Proinsulin I and Products (or ExpectedProducts) from Enzyme Cocktail Cleavage of Each Conjugate Product(Expected Products) FIG. Proinsulin I Mono A Insulin 16 Proinsulin IMono B (Lys-hexyl-PEG7-oligomer-conjugated leader peptide) — ProinsulinI Di Lys^(B29)-Hexyl-PEG7-Oligomer-Conjugated Insulin 15 Proinsulin ITri (Lys^(B29)-Hexyl-PEG7-Oligomer-Conjugated Insulin and —Lys-hexyl-PEG7-oligomer-conjugated C-peptide)

Example 14 Trypsin Cleavage of Isolated Conjugates of Proinsulin I

Each conjugate (Proinsulin I Mono A, Mono B, Di, or Tri) that wasisolated using the procedure described in Example 12 is dissolved in 100mM Tris-HCl Buffer, pH 7.6, and the resulting solution is analyzed byHPLC to determine the polypeptide concentration. A solution of trypsin(TPCK treated; from bovine pancreas) is prepared in 100 mM Tris-HCl 15Buffer, pH 7.6. Each conjugate (300 mmol) is then allowed to react withtrypsin (1 mmol). After 20 minutes, the reaction is quenched by additionof 1% trifluoroacetic acid in acetonitrile. The products of the reactionare isolated and analyzed by HPLC retention time and mass spectralanalysis to determine identity. The expected products are Insulin(Arg³¹)or Lys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin(Arg³¹) illustratedin Table 2.

TABLE 2 Conjugate Expected Products Proinsulin I Mono A Insulin(Arg³¹)and Lys-hexyl-PEG7-oligomer conjugated C-peptide Proinsulin I Mono BLys^(B29)-hexyl-PEG7-oligomer-conjugated insulin(Arg³¹) and C-peptideProinsulin I Di Lys^(B29)-hexyl-PEG7-oligomer-conjugated insulin(Arg³¹)and Lys-hexyl-PEG7-oligomer conjugated C-peptide Proinsulin I TriLys^(B29)-hexyl-PEG7-oligomer-conjugated insulin(Arg³¹) andLys-hexyl-PEG7-oligomer-conjugated C-peptide and Lys-hexyl-PEG7-oligomerconjugated leader peptide

Example 15 Carboxypeptidase B Cleavage of Trypsin Cleavage ProductMixture

An aliquot of the reaction mixture containingLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin(Arg³¹) (300 mmol) (fromExample 14) in 100 mM Tris-HCl buffer, pH 7.6, is removed. A solution ofcarboxypeptidase B (from porcine pancreas) is prepared in 100 mM TrisHCl buffer, pH 7.6. Carboxypeptidase B (1 mmol) is added to the reactionmixture. The reaction is allowed to continue for 15 hours, and then isquenched with addition of 1% trifluoroacetic acid in acetonitrile. Theexpected products of each reaction are illustrated in Table 3.

TABLE 3 Conjugate Expected Products Proinsulin I Mono A Insulin andLys-hexyl-PEG7-oligomer conjugated C-peptide Proinsulin I Mono BLys^(B29)-hexyl-PEG7-oligomer-conjugated insulin and C-peptideProinsulin I Di Lys^(B29)-hexyl-PEG7-oligomer-conjugated insulin andLys-hexyl-PEG7-oligomer conjugated C-peptide Proinsulin I TriLys^(B29)-hexyl-PEG7-oligomer-conjugated insulin andLys-hexyl-PEG7-oligomer-conjugated C-peptide and Lys-hexyl-PEG7-oligomerconjugated leader peptide

Example 16

Preparation of Lys^(B29)-Oligomer Conjugated Insulin

(a) Conjugation of Recombinant Proinsulin II. Recombinant Proinsulin II(NW 11,133 Daltons) was obtained from Itoham Foods, Inc. of IbarakiPref, Japan. The Recombinant Proinsulin II had a leader peptide and aC-peptide that were each devoid of Lysine residues. A 2.55×10-3 mmolportion of recombinant Proinsulin II was dissolved in 10 mL of DMSO. Tothe solution was added 355 μL of triethylamine. The resulting solutionwas allowed to stir for 5 minutes, and then a solution of activatedmethylpolyethylene glycol ((PEGn)hexyl oligomer) (n=7±3 or n=7) (5.10×10⁻³ mmol) in acetonitrile was added. The course of the reaction wasmonitored by HPLC. After the reaction appeared to be complete, it wasquenched by addition of 3.7 mL of 5% aqueous trifluoroacetic acidsolution The reaction mixture was then processed and exchanged into 100mM Tris-HCl Buffer, pH 7.6. The HPLC profile of the oligomer-conjugatedrecombinant Proinsulin II product mixture is shown in FIG. 2.

(b) Enzyme Cocktail Cleavage of Oligomer-Conjugated RecombinantProinsulin II.

The Tris-HCl solution of the product mixture from Example 16(a) wasanalyzed by HPLC to determine the polypeptide concentration therein. Asolution of trypsin (TPCK treated; from bovine pancreas) was prepared in100 mM Tris-HCl Buffer, pH 7.6. A solution of carboxypeptidase B (fromporcine pancreas) was prepared in 100 mM Tris-HCl Buffer, pH 7.6. Theproduct mixture (0.399 μmol/mL) was allowed to react with trypsin(5.57×10⁻⁴ μmol/mL) and carboxypeptidase B (1.82×10⁻⁴ μmol/mL). After 30minutes, the reaction was quenched by addition of 550 μL of 1%trifluoroacetic acid in acetonitrile; The major products were identifiedby HPLC retention time (relative to that of known reference standards)and mass spectral analysis. Insulin (23%) andLys^(B29)-hexyl-PEGn-oligomer-conjugated Insulin (60%) and other (17%)were thus obtained (FIGS. 9-10).

Example 17 Isolation of the Products of Oligomer-Conjugation ofRecombinant Proinsulin II

Each major product from the conjugation reaction described in Example16(a) was isolated using reversed-phase HPLC. A column (1.0 cm. i.d.×25cm. length) was packed with a commercially available C18 stationaryphase known to be useful for the resolution of polypeptides andproteins, and then was incorporated into an HPLC system. The system wasequilibrated with elution buffer that was a mixture of 75% mobile phaseA (H₂O with 0.1% trifluoroacetic acid) and 25% mobile phase B(acetonitrile with 0.1% trifluoroacetic acid) that was delivered at aflow rate of 5 mL/min. The Tris-HCl solution of the product mixture fromExample 16(a) was applied to the column, and the major products wereseparated and eluted using a gradient elution in which the compositionof the elution buffer was changed from 25% mobile phase B to 35% mobilephase B over 120 minutes. Each of the fractions that were collected wasanalyzed by HPLC to determine the identity and purity of the productcontained therein. Common fractions of each product (Proinsulin IImonoconjugate (“Proinsulin II Mono”) and diconjugate (“Proinsulin IIDi”) were then pooled, and the solvent was removed by rotaryevaporation. The identity and purity of each product were determined byHPLC and mass spectrometric analyses (FIGS. 2-4).

Example 18 Enzyme Cocktail Cleavage of Isolated Conjugates ofRecombinant Proinsulin II

Each Proinsulin II conjugate (Proinsulin II Mono, Di or Tri) that wasisolated using the procedure described in Example 17 was dissolved in100 mM Tris-HCl Buffer, pH 7.6, and an aliquot of the solution wasanalyzed by HPLC to determine the polypeptide concentration therein. Asolution of trypsin (TPCK treated; from bovine pancreas) was prepared in100 mM Tris-HCl Buffer, pH 7.6. A solution of carboxypeptidase B (fromporcine pancreas) was prepared in 100 mM Tris-HCl Buffer, pH 7.6. Theconjugate (0.127 μmol/mL) was allowed to react with trypsin (1.77×10⁻⁴umol/mL) and carboxypeptidase B (5.77×10⁻⁵ μmol/mL). After 30 minutes,the reaction was quenched by addition of 250 μL of 1% trifluoroaceticacid in acetonitrile. Isolation of the major products followed byidentification by HPLC retention time against reference standards andmass spectral analysis showed that Insulin or B-29 acylatedInsulin-hexyl-PEG7 were produced in the reaction. The products andyields of each reaction are illustrated in Table 4.

TABLE 4 Conjugate Expected Products Yield Proinsulin II Mono Insulin 15%Lys^(B29)-hexyl-PEGn-oligomer 85% conjugated insulin Proinsulin II DiLys^(B29)-hexyl-PEGn-oligomer- 92% conjugated insulin

Example 19 Trypsin Cleavage of Isolated Conjugates of Proinsulin II

Each conjugate (Proinsulin II Mono, Di or Tri) from Example 17 wasdissolved in 100 mM Tris-HCl Buffer, pH 7.6, and the resulting solutionwas analyzed by HPLC to determine the polypeptide concentration therein.A solution of trypsin (TPCK treated; from bovine pancreas) was preparedin 100 mM Tris-HCl Buffer, pH 7.6. Each conjugate (0.127 μmol/mL) wasthen allowed to react with trypsin (4.23×10⁻⁴ μmol/mL). After 20minutes, reaction was quenched by addition of 250 μL of 1%trifluoroacetic acid in acetonitrile. Isolation of the major productsfollowed by identification by HPLC retention time and mass spectralanalysis showed that Insulin(Arg³¹) orLys^(B29)-hexyl-PEGn-oligomer-conjugated Insulin(Arg³¹) was produced inthe reaction. The products and yields of each reaction are illustratedin Table 5.

Example 20 Carboxypeptidase B Cleavage of Trypsin Cleavage Mixture

An aliquot of the reaction mixture ofLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin(Arg³¹) (3.10×10⁻5 mmol)from Example 19 was removed. A solution of carboxypeptidase B (fromporcine pancreas) was prepared in 100 mM Tris-HCl buffer pH 7.6.Carboxypeptidase B (1.03×10⁻⁷) was added to the reaction mixture.Reaction was allowed to continue for 15 hours, and then was quenchedwith addition of 1% trifluoroacetic acid in acetonitrile. Afterprocessing, the products of the reaction were analyzed by HPLC.Retention time and mass spectral analysis were used to determineidentity. Insulin (23%) and Lys^(B29)-hexyl-PEGn-oligomer-conjugatedinsulin (60%) (FIGS. 5, 7-8) were produced from the reaction ofProinsulin II monoconjugate. The expected products of the Proinsulin IIdiconjugate reaction are illustrated in Table 6.

TABLE 6 Conjugate Products (Expected Products) Yield Proinsulin IIInsulin and 23% Mono Lys^(B29)-hexyl-PEGn-oligomer-conjugated Insulin60% Proinsulin II Di (Lys^(B29)-Hexyl-PEGn-oligomer-conjugated Insulin)

Example 21 Preparation of Lys^(B29)-Oligomer-Conjugated Insulin

(a) Conjugation of Natural Human Proinsulin. Natural human proinsulin(Sigma Chemical Co.) (3.20×10⁻⁴ mmol) is dissolved in 5 mL of DMSO. Tothe solution is added 45 μl of triethylamine. The solution is allowed tostir for 5 minutes before a solution of activated PEG7-hexyl oligomer(6.4×10⁻⁴ mmol) in acetonitrile is added. After the reaction hasprogressed such that HPLC analysis indicates that the proinsulin hasbeen consumed (or the concentration of proinsulin is no longerdecreasing), the reaction is quenched by addition of 0.5 mL of 5%aqueous trifluoroacetic acid solution. The reaction mixture is thenprocessed and exchanged into 100 mM Tris-HCl Buffer, pH 7.6.

(b) Enzyme Cocktail Cleavage of Oliomer-Conjugated Natural Proinsulin.An aliquot of the Tris-HCl solution of the product mixture from Example21(a) is analyzed by HPLC to determine the polypeptide concentrationtherein A solution of trypsin (TPCK treated; from bovine pancreas) isprepared in 100 mM Tris-HCl Buffer, pH 7.6. A solution ofcarboxypeptidase B (from porcine pancreas) is prepared in 100 mMTris-HCl Buffer, pH 7.6. The crude mixture (1 mol eq.) is then allowedto react with trypsin (1.39×10⁻³ mol eq) and carboxypeptidase B(4.56×10⁻⁴ mol eq.). After 30 minutes, the reaction is quenched byaddition of 1% trifluoroacetic acid in acetonitrile. The product mixtureof the reaction is processed and analyzed by HPLC. Retention time(versus that of reference standards) and mass spectral analysis are usedto determine identity. The expected products of the reaction are Insulinand Lys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin.

Example 22 Isolation of the Products of Conjugation of Natural HumanProinsulin

Each major product obtained from the conjugation reaction described inExample 11(a) is isolated using reversed-phase HPLC. A column (1.0 cm.i.d.×25 cm. length) is packed with a commercially available C18stationary phase known to be useful for the resolution of polypeptidesand proteins, and then is incorporated into an HPLC system. The systemis equilibrated with elution buffer that comprises a mixture of 75%mobile phase A (H2O with 0.1% trifluoroacetic acid) and 25% mobile phaseB (acetonitrile with 0.1% trifluoroacetic acid). The Tris-HCl solutionof the product mixture from Example 21(a) is applied to the column, andthe major products are separated and eluted using a gradient elution inwhich the percentage of the acetonitrile component is increased from25%-35% over 120 minutes. Fractions are collected and analyzed by HPLCto determine the identity and purity of the product therein. Commonfractions of each product are pooled, and the solvent is removed byrotary evaporation. The identity and purity of each product peak aredetermined by HPLC and mass spectrometry. The expected products consistof 2 human Proinsulin monoconjugates, 1 human Proinsulin diconjugate and1 human Proinsulin triconjugate.

Example 23 Enzyme Cocktail Cleavage of Isolated Conjugates of NaturalHuman Proinsulin

Each conjugate that is obtained using the procedure described in Example22 is dissolved in 100 mM Tris-HCl Buffer, pH 7.6, and the resultingsolution is analyzed by HPLC to determine the polypeptide concentrationtherein. A solution of trypsin (TPCK treated; from bovine pancreas) isprepared in 100 mM Tris-HCl Buffer, pH 7.6. A solution ofcarboxypeptidase B (from porcine pancreas) is prepared in 100 mMTris-HCl Buffer, pH 7.6. The crude mixture (1 mol eq.) is then allowedto react with trypsin (1.39×10⁻³ mol eq.) and carboxypeptidase B(4.56×10⁻⁴ mol eq.). After 30 minutes, the reaction is quenched byaddition of 1% trifluoroacetic acid in acetonitrile. The products areprocessed and analyzed by HPLC. Retention time (compared to that ofreference standards) and mass spectral analysis are used to determineidentity. The expected products of the reaction are Insulin orLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin.

Example 24 Trypsin Cleavage of Isolated Conjugates of Natural HumanProinsulin

Each conjugate that is obtained as described in Example 22 is dissolvedin 100 mM Tris-HCl Buffer, pH 7.6, and the resulting solution isanalyzed by HPLC to determine the polypeptide concentration therein. Asolution of trypsin (TPCK treated; from bovine pancreas) is prepared in100 mM Tris-HCl Buffer, pH 7.6. The conjugate (300 mol eq.) is thenallowed to react with trypsin (1 mol eq). After 20 minutes, reaction isquenched by addition of 1% trifluoroacetic acid in acetonitrile. Theproducts are processed and analyzed by HPLC. Retention time and massspectrometry are used to determine identity. The expected products ofthe reaction are Insulin(Arg³¹) orLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin(Arg³¹).

Example 25 Carboxypeptidase B Cleavage of Trypsin Cleavage Mixture

An aliquot of the reaction mixture ofLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin(Arg³¹) (300 mmol) fromExample 24 is removed. A solution of carboxypeptidase B (from porcinepancreas) is prepared in 100 mM Tris-HCl buffer, pH 7.6.Carboxypeptidase B(1 mmol) is added to the reaction mixture. Reaction isallowed to continue for 15 hours, and then is quenched with addition of1% trifluoroacetic acid in acetonitrile. The products are processed andanalyzed by HPLC. Retention time and mass spectral analysis are used todetermine identity. The expected products are Insulin orLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin.

Example 26 Optimized Preparation of Lys^(B29)-Oligomer-ConjugatedInsulin

Analysis of the experimental data from Example 11 indicated thatLys^(B29)-hexyl-PEG7-oligomer-conjugated Insulin andLys-hexyl-PEG7-oligomer-conjugated C-peptide could be obtained in highyield and purity by (a) acylating the ε-amino group of all lysineresidues that are present on a proinsulin raw material, and (b) cleavingthe resulting, filly oligomer-conjugated proinsulin with an enzymecocktail made up of trypsin and carboxypeptidase B. Experimentalconfirmation of this hypothesis was obtained as follows.

(a) Conjugation of Recombinant Proinsulin I. Recombinant Proinsulin I(MW 10,642 Daltons) is obtained from Biobras, Belo Horizonte, Brazil. A2.32×10⁻³ mmol portion of proinsulin I is dissolved in 10 mL of DMSO. Tothe solution is added 324 μL of triethylamine. The resulting solution isallowed to stir for 5 minutes, and then a solution of activatedmethylheptaethylene glycol(PEG7)-hexyl oligomer (4-6 mol eq.; sufficientto covert all Proinsulin I to the triconjugate) in acetonitrile isadded. The course of the conjugation (acylation) reaction is monitoredby HPLC. When reaction appears to be complete (i.e., no unconjugatedProinsulin I is observed by HPLC), it is quenched by addition of 3.54 mLof 5% aqueous trifluoroacetic acid solution. The reaction mixture isthen processed and exchanged into 100 mM Tris-HCl Buffer, pH 7.6. TheHPLC profile of the product mixture, oligomer-conjugated recombinantProinsulin I, is expected to show peaks corresponding to triconjugate(all Lys and N-terminus conjugated) and diconjugate only.

(b) Enzyme Cocktail Cleavage of Oligomer-Conjugated RecombinantProinsulin I.

An aliquot of the Tris-HCl solution of the product mixture from Example16(a) is analyzed by HPLC to determine the polypeptide concentration. Asolution of trypsin (TPCK treated; from bovine pancreas) is prepared in100 mM Tris-HCl Buffer, pH 7.6. A solution of carboxypeptidase B (fromporcine pancreas) is prepared in 100 mM Tris-HCl Buffer, pH 7.6. Theproduct mixture from Example 16(a) (0.424 μmol/mL) is then allowed toreact with trypsin (5.97×10⁻⁴ μmol/mL) and carboxypeptidase B (1.93×10⁻⁴μmol/mL). After 30 minutes, the reaction is quenched by the addition of1.58 mL of 1% trifluoroacetic acid in acetonitrile. The major productsare identified by HPLC retention time (relative to the retention timesof known reference standards) and mass spectral analysis.Lys^(B29)-Hexyl-PEG7-Oligomer-Conjugated Insulin, the only insulinconjugate that is present, is expected to be obtained in near 95% yield.Lys¹-Hexyl-PEG7-Oligomer-Conjugated C-peptide is also obtained in nearquantitative yield.

The present invention has been described herein with reference to itspreferred embodiments. These embodiments do not serve to limit theinvention, but are set forth for illustrative purposes. The scope of theinvention is defined by the claims that follow.

1. A method of synthesizing an insulin polypeptide-oligomer conjugatecomprising: contacting a proinsulin polypeptide comprising an insulinpolypeptide coupled to one or more peptides by peptide bond(s) capableof being cleaved to yield the insulin polypeptide with an oligomercomprising a hydrophilic moiety and a lipophilic moiety under conditionssufficient to couple the oligomer to the insulin polypeptide portion ofthe proinsulin polypeptide and provide a proinsulin polypeptide-oligomerconjugate; and cleaving the one or more peptides from the proinsulinpolypeptide-oligomer conjugate to provide the insulinpolypeptide-oligomer conjugate.
 2. The method according to claim 1,wherein the contacting of the proinsulin polypeptide with the oligomercomprises: contacting the oligomer with an activating agent underconditions sufficient to provide an activated oligomer capable ofcoupling to a nucleophilic functionality on the proinsulin polypeptide;and contacting the activated oligomer with the proinsulin polypeptideunder conditions sufficient to provide the proinsulinpolypeptide-oligomer conjugate.
 3. The method according to claim 2,wherein the contacting of the oligomer with the activating agent and thecontacting of the activated oligomer with the proinsulin polypeptide isperformed in situ.
 4. The method according to claim 2, wherein the molarratio of activated oligomer to proinsulin polypeptide is greater thanabout 1:1.
 5. The method according to claim 2, wherein the molar ratioof activated oligomer to proinsulin polypeptide is greater than about3:1.
 6. The method according to claim 2, wherein the molar ratio ofactivated oligomer to proinsulin polypeptide is greater than about 4:1.7. The method according to claim 6, wherein the yield of insulinpolypeptide-oligomer conjugate is greater than 75 percent.
 8. The methodaccording to claim 6, wherein the yield of insulin polypeptide-oligomerconjugate is greater than 80 percent.
 9. The method according to claim6, wherein the yield of insulin polypeptide-oligomer conjugate isgreater than 85 percent.
 10. The method according to claim 6, whereinthe yield of insulin polypeptide-oligomer conjugate is greater thanabout 90 percent.
 11. The method according to claim 1, wherein the yieldof insulin polypeptide-oligomer conjugate is greater than 75 percent.12. The method according to claim 1, wherein the yield of insulinpolypeptide-oligomer conjugate is greater than 80 percent.
 13. Themethod according to claim 1, wherein the yield of insulinpolypeptide-oligomer conjugate is greater than 85 percent.
 14. Themethod according to claim 1, wherein the yield of insulinpolypeptide-oligomer conjugate is greater than about 90 percent.
 15. Themethod according to claim 1, wherein the yield of insulinpolypeptide-oligomer conjugate is greater than about 95 percent.
 16. Themethod according to claim 1, wherein the insulin polypeptide has anA-chain polypeptide and a B-chain polypeptide, and wherein the one ormore peptides comprise a connecting peptide coupled at a first end tothe C-terminus of the B-chain polypeptide and coupled at a second end tothe N-terminus of the A-chain polypeptide.
 17. The method according toclaim 16, wherein the connecting peptide is a C-peptide polypeptide. 18.The method according to claim 16, wherein the connecting peptide isC-peptide.
 19. The method according to claim 16, wherein the connectingpeptide is devoid of lysine residues.
 20. The method according to claim16, wherein the one or more peptides further comprise a leader peptidecoupled to the N-terminus of the B-chain polypeptide.
 21. The methodaccording to claim 20, wherein the leader peptide is devoid of lysineresidues.
 22. The method according to claim 1, wherein the insulinpolypeptide has an A-chain polypeptide and a B-chain polypeptide, andwherein the one or more peptides is a connecting peptide coupled at afirst end to the C-terminus of the B-chain polypeptide and at a secondend to the N-terminus of the A-chain polypeptide.
 23. The methodaccording to claim 1, wherein the insulin polypeptide has an A-chainpolypeptide and a B-chain polypeptide, and wherein the one or morepeptides is a connecting peptide coupled at a first end to theC-terminus of the B-chain polypeptide and at a second end to theN-terminus of the A-chain polypeptide, and a leader peptide coupled tothe N-terminus of the B-chain polypeptide.
 24. The method according toclaim 1, wherein the proinsulin polypeptide is proinsulin.
 25. Themethod according to claim 1, wherein the proinsulin polypeptide isproinsulin coupled at the N-terminus of the B-chain to a leader peptideby a peptide bond that is cleavable.
 26. The method according to claim1, wherein the insulin polypeptide is insulin.
 27. The method accordingto claim 26, wherein the oligomer is coupled to the lysine at the B29position of the insulin.
 28. The method according to claim 1, whereinthe insulin polypeptide is an insulin analog selected from the groupconsisting of GlyA21 insulin, human; GlyA21 GlnB3 insulin, human; AlaA21insulin, human; AlaA21 GlnB3 insulin, human; GlnB3 insulin, human;GlnB30 insulin, human; GlyA21 GluB30 insulin, human; GlyA21 GlnB3 GluB30insulin, human; GlnB3 GluB30 insulin, human; AspB28 insulin, human;LysB28 insulin, human; LeuB28 insulin, human; ValB28 insulin, human;AlaB28 insulin, human; AspB28 ProB29 insulin, human; LysB28 ProB29insulin, human; LeuB28 ProB29 insulin, human; ValB28 ProB29 insulin,human; AlaB28 ProB29 insulin, human.
 29. The method according to claim1, wherein the insulin polypeptide-oligomer conjugate is amphiphilicallybalanced.
 30. The method according to claim 1, wherein the oligomer ispresent as a substantially monodispersed mixture.
 31. The methodaccording to claim 1, wherein the oligomer is present as a monodispersedmixture.
 32. The method according to claim 1, wherein the hydrophilicmoiety is a polyalkylene glycol moiety.
 33. The method according toclaim 32, wherein the polyalkylene glycol moiety is a polyethyleneglycol moiety.
 34. The method according to claim 32, wherein thepolyalkylene glycol moiety has between 1 and 50 polyalkylene glycolsubunits.
 35. The method according to claim 32, wherein the polyalkyleneglycol moiety has between 3 and 50 polyalkylene glycol subunits.
 36. Themethod according to claim 32, wherein the polyalkylene glycol moiety hasbetween 2 and 10 polyalkylene glycol subunits.
 37. The method accordingto claim 32, wherein the polyalkylene glycol moiety has between 4 and 10polyalkylene glycol subunits.
 38. The method according to claim 32,wherein the polyalkylene glycol moiety has at least 2 polyalkyleneglycol subunits.
 39. The method according to claim 1, wherein thelipophilic moiety is an alkyl or fatty acid moiety.
 40. The methodaccording to claim 1, wherein the lipophilic moiety has between 1 and 28carbon atoms.
 41. The method according to claim 1, wherein thelipophilic moiety has between 2 and 24 carbon atoms.
 42. The methodaccording to claim 1, wherein the lipophilic moiety has between 3 and 18carbon atoms.
 43. The method according to claim 1, wherein thelipophilic moiety has between 4 and 12 carbon atoms.
 44. The methodaccording to claim 1, wherein the lipophilic moiety has between 5 and 7carbon atoms.
 45. The method according to claim 1, wherein thelipophilic moiety has between 4 and 14 carbon atoms.
 46. The methodaccording to claim 1, wherein the cleaving of the one or more peptidesfrom the proinsulin polypeptide-oligomer conjugate comprises contactingthe proinsulin polypeptide-oligomer conjugate with one or more enzymesthat are capable of cleaving the bond(s) between the one or morepeptides and the insulin polypeptide under conditions sufficient tocleave the one or more peptides from the proinsulin polypeptide-oligomerconjugate.
 47. The method according to claim 46, wherein the one or moreenzymes are selected from the group consisting of trypsin, carboxypeptidase B, and mixtures thereof.
 48. The method according to claim 16,wherein the connecting peptide has a terminal amino acid residue at thefirst end, and wherein the cleaving of the connecting peptide from theproinsulin polypeptide-oligomer conjugate comprises: contacting theproinsulin polypeptide-oligomer conjugate with a first enzyme underconditions sufficient to provide a terminal amino acid residue-insulinpolypeptide-oligomer conjugate; and contacting the terminal amino acidresidue-insulin polypeptide-oligomer conjugate with a second enzymeunder conditions sufficient to provide the insulin polypeptide-oligomerconjugate.
 49. The method according to claim 48, wherein the terminalamino acid residue is an arginine residue.
 50. The method according toclaim 49, wherein the insulin polypeptide is insulin, and wherein theconnecting peptide is human C-peptide.
 51. The method according to claim48, wherein the contacting of the proinsulin polypeptide-oligomerconjugate with a first enzyme and the contacting of the terminal aminoacid residue-insulin polypeptide-oligomer conjugate with a second enzymeoccur substantially concurrently.
 52. The method according to claim 51,wherein the first enzyme and the second enzyme are provided in a mixturecomprising the first enzyme and the second enzyme.
 53. The methodaccording to claim 48, wherein the first enzyme is trypsin, and whereinthe second enzyme is carboxy peptidase B.
 54. A method of synthesizingan insulin polypeptide-acyl oligomer conjugate comprising enzymaticallycleaving one or more peptides from a proinsulin polypeptide-acyloligomer conjugate to provide the insulin polypeptide-acyl oligomerconjugate.
 55. The method according to claim 54, wherein the insulinpolypeptide has an A-chain polypeptide and a B-chain polypeptide, andwherein the one or more peptides comprise a connecting peptide coupledat a first end to the C-terminus of the B-chain polypeptide and coupledat a second end to the N-terminus of the A-chain polypeptide.
 56. Themethod according to claim 55, wherein the connecting peptide is aC-peptide polypeptide.
 57. The method according to claim 55, wherein theconnecting peptide is C-peptide.
 58. The method according to claim 55,wherein the connecting peptide is devoid of lysine residues.
 59. Themethod according to claim 55, wherein the one or more peptides furthercomprise a leader peptide coupled to the N-terminus of the B-chainpolypeptide.
 60. The method according to claim 55, wherein the leaderpeptide is devoid of lysine residues.
 61. The method according to claim54, wherein the proinsulin polypeptide is proinsulin.
 62. The methodaccording to claim 54, wherein the proinsulin polypeptide is proinsulincoupled at its N-terminus to a leader peptide by a peptide bond that iscleavable.
 63. The method according to claim 54, wherein the insulinpolypeptide is insulin.
 64. The method according to claim 63, whereinthe acyl oligomer is coupled to the lysine at the B29 position of theinsulin.
 65. The method according to claim 54, wherein the insulinpolypeptide-acyl oligomer conjugate is amphiphilically balanced.
 66. Themethod according to claim 54, wherein the acyl oligomer portion of theinsulin polypeptide-acyl oligomer conjugate comprises a hydrophilicmoiety and a lipophilic moiety.
 67. The method according to claim 66,wherein the hydrophilic moiety is a polyethylene glycol moiety.
 68. Themethod according to claim 67, wherein the polyethylene glycol moiety hasbetween 1 and 50 polyethylene glycol subunits.
 69. The method accordingto claim 67, wherein the polyethylene glycol moiety has between 3 and 50polyethylene glycol subunits.
 70. The method according to claim 67,wherein the polyethylene glycol moiety has between 2 and 10 polyethyleneglycol subunits.
 71. The method according to claim 67, wherein thepolyethylene glycol moiety has between 4 and 10 polyethylene glycolsubunits.
 72. The method according to claim 67, wherein the polyethyleneglycol moiety has at least 2 polyethylene glycol subunits.
 73. Themethod according to claim 66, wherein the lipophilic moiety is an alkylor a fatty acid moiety.
 74. The method according to claim 73, whereinthe lipophilic moiety has between 1 and 28 carbon atoms.
 75. The methodaccording to claim 73, wherein the lipophilic moiety has between 2 and24 carbon atoms.
 76. The method according to claim 73, wherein thelipophilic moiety has between 3 and 18 carbon atoms.
 77. The methodaccording to claim 73, wherein the lipophilic moiety has between 4 and12 carbon atoms.
 78. The method according to claim 73, wherein thelipophilic moiety has between 5 and 7 carbon atoms.
 79. The methodaccording to claim 73, wherein the lipophilic moiety has between 4 and14 carbon atoms.
 80. The method according to claim 54, wherein theenzymatically cleaving of the one or more peptides from the proinsulinpolypeptide-acyl oligomer conjugate comprises contacting the proinsulinpolypeptide-oligomer conjugate with one or more enzymes that are capableof cleaving the bond(s) between the one or more peptides and the insulinpolypeptide under conditions sufficient to cleave the one or morepeptides from the proinsulin polypeptide-oligomer conjugate.
 81. Themethod according to claim 80, wherein the one or more enzymes areselected from the group consisting of trypsin, carboxy peptidase B, andmixtures thereof.
 82. The method according to claim 55, wherein theconnecting peptide has a terminal amino acid residue at the first end,and wherein the enzymatically cleaving of the connecting peptide fromthe proinsulin-acyl oligomer conjugate comprises: contacting theproinsulin polypeptide-acyl oligomer conjugate with a first enzyme underconditions sufficient to provide a terminal amino acid residue-insulinpolypeptide-oligomer conjugate; and contacting the terminal amino acidresidue-insulin polypeptide-acyl oligomer conjugate with a second enzymeunder conditions sufficient to provide the insulin-acyl oligomerconjugate.
 83. The method according to claim 82, wherein the terminalamino acid residue is an arginine residue.
 84. The method according toclaim 83, wherein the insulin polypeptide is insulin, and wherein theconnecting peptide is C-human peptide.
 85. The method according to claim82, wherein the contacting of the proinsulin-oligomer conjugate with afirst enzyme and the contacting of the terminal amino acidresidue-insulin polypeptide-acyl oligomer conjugate with a second enzymeoccur substantially concurrently.
 86. The method according to claim 82,wherein the first enzyme and the second enzyme are provided in a mixturecomprising the first enzyme and the second enzyme.
 87. The methodaccording to claim 82, wherein the first enzyme is trypsin, and whereinthe second enzyme is carboxy peptidase B.
 88. A method of synthesizing aproinsulin polypeptide-oligomer conjugate comprising contacting aproinsulin polypeptide with an oligomer comprising a hydrophilic moietyand a lipophilic moiety under conditions sufficient to provide theproinsulin polypeptide-oligomer conjugate.
 89. The method according toclaim 88, wherein the proinsulin polypeptide comprises an insulinpolypeptide having an A-chain polypeptide, a B-chain polypeptide, and aconnecting peptide coupled at a first end to the C-terminus of theB-chain polypeptide and coupled at a second end to the N-terminus of theA-chain polypeptide.
 90. The method according to claim 89, wherein theconnecting peptide is a C-peptide polypeptide.
 91. The method accordingto claim 89, wherein the connecting peptide is C-peptide.
 92. The methodaccording to claim 89, wherein the connecting peptide is devoid oflysine residues.
 93. The method according to claim 89, wherein theproinsulin polypeptide further comprises a leader peptide coupled to theN-terminus of the B-chain polypeptide.
 94. The method according to claim93, wherein the leader peptide is devoid of lysine residues.
 95. Themethod according to claim 88, wherein the proinsulin polypeptidecomprises an insulin polypeptide having an A-chain polypeptide and aB-chain polypeptide, a connecting peptide coupled at a first end to theC-terminus of the B-chain polypeptide and at a second end to theN-terminus of the A-chain polypeptide, and a leader peptide coupled tothe N-terminus of the B-chain polypeptide.
 96. The method according toclaim 95, wherein the insulin polypeptide is insulin.
 97. The methodaccording to claim 96, wherein the oligomer is coupled to the lysine atthe B29 position of the insulin.
 98. The method according to claim 88,wherein the proinsulin polypeptide is proinsulin.
 99. The methodaccording to claim 88, wherein the insulin polypeptide-oligomerconjugate is amphiphilically balanced.
 100. The method according toclaim 88, wherein the oligomer is present as a substantiallymonodispersed mixture.
 101. The method according to claim 88, whereinthe oligomer is present as a monodispersed mixture.
 102. The methodaccording to claim 88, wherein the hydrophilic moiety is a polyalkyleneglycol moiety.
 103. The method according to claim 102, wherein thepolyalkylene glycol moiety is a polyethylene glycol moiety.
 104. Themethod according to claim 102, wherein the polyalkylene glycol moietyhas between 1 and 50 polyalkylene glycol subunits.
 105. The methodaccording to claim 102, wherein the polyalkylene glycol moiety hasbetween 3 and 50 polyalkylene glycol subunits.
 106. The method accordingto claim 102, wherein the polyalkylene glycol moiety has between 2 and10 polyalkylene glycol subunits.
 107. The method according to claim 102,wherein the polyalkylene glycol moiety has between 4 and 10 polyalkyleneglycol subunits.
 108. The method according to claim 102, wherein thepolyalkylene glycol moiety has at least 2 polyalkylene glycol subunits.109. The method according to claim 88, wherein the lipophilic moiety isan alkyl or fatty acid moiety.
 110. The method according to claim 88,wherein the lipophilic moiety has between 1 and 28 carbon atoms. 111.The method according to claim 88, wherein the lipophilic moiety hasbetween 2 and 24 carbon atoms.
 112. The method according to claim 88,wherein the lipophilic moiety has between 3 and 18 carbon atoms. 113.The method according to claim 88, wherein the lipophilic moiety hasbetween 4 and 12 carbon atoms.
 114. The method according to claim 88,wherein the lipophilic moiety has between 5 and 7 carbon atoms.
 115. Themethod according to claim 88, wherein the lipophilic moiety has between4 and 14 carbon atoms.